Nucleobase editors comprising nucleic acid programmable DNA binding proteins

RELATED APPLICATIONS

0001This application claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent applications, U.S. Ser. No. 62/551,951, filed Aug. 30, 2017; U.S. Ser. No. 62/511,934, filed May 26, 2017; U.S. Ser. No. 62/490,587, filed Apr. 26, 2017; and U.S. Ser. No. 62/475,830, filed Mar. 23, 2017, each of which are incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

0002The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 7, 2020, is named H082470257US04-SEQ-CHB and is 1,336,633 bytes in size.

BACKGROUND OF THE INVENTION

0003Targeted editing of nucleic acid sequences, for example, the targeted cleavage or the targeted introduction of a specific modification into genomic DNA, is a highly promising approach for the study of gene function and also has the potential to provide new therapies for human genetic diseases.¹An ideal nucleic acid editing technology possesses three characteristics: (1) high efficiency of installing the desired modification; (2) minimal off-target activity; and (3) the ability to be programmed to edit precisely any site in a given nucleic acid, e.g., any site within the human genome.²Current genome engineering tools, including engineered zinc finger nucleases (ZFNs),³transcription activator like effector nucleases (TALENs),⁴and most recently, the RNA-guided DNA endonuclease Cas9,⁵effect sequence-specific DNA cleavage in a genome. This programmable cleavage can result in mutation of the DNA at the cleavage site via non-homologous end joining (NHEJ) or replacement of the DNA surrounding the cleavage site via homology-directed repair (HDR).^6,7

0004One drawback to the current technologies is that both NHEJ and HDR are stochastic processes that typically result in modest gene editing efficiencies as well as unwanted gene alterations that can compete with the desired alteration.⁸Since many genetic diseases in principle can be treated by effecting a specific nucleotide change at a specific location in the genome (for example, a C to T change in a specific codon of a gene associated with a disease),⁹the development of a programmable way to achieve such precision gene editing would represent both a powerful new research tool, as well as a potential new approach to gene editing-based human therapeutics.

SUMMARY OF THE INVENTION

0005Nucleic acid programmable DNA binding proteins (napDNAbp), such as the clustered regularly interspaced short palindromic repeat (CRISPR) system is a recently discovered prokaryotic adaptive immune system¹⁰that has been modified to enable robust and general genome engineering in a variety of organisms and cell lines.¹¹CRISPR-Cas (CRISPR associated) systems are protein-RNA complexes that use an RNA molecule (sgRNA) as a guide to localize the complex to a target DNA sequence via base-pairing.¹²In the natural systems, a Cas protein then acts as an endonuclease to cleave the targeted DNA sequence.¹³The target DNA sequence must be both complementary to the sgRNA, and also contain a “protospacer-adjacent motif” (PAM) at the 3′-end of the complementary region in order for the system to function.¹⁴

0006Among the known Cas proteins, <i>S. pyogenes </i>Cas9 has been mostly widely used as a tool for genome engineering.¹⁵This Cas9 protein is a large, multi-domain protein containing two distinct nuclease domains. Point mutations can be introduced into Cas9 to abolish nuclease activity, resulting in a dead Cas9 (dCas9) that still retains its ability to bind DNA in a sgRNA-programmed manner.¹⁶In principle, when fused to another protein or domain, dCas9 can target that protein or domain to virtually any DNA sequence simply by co-expression with an appropriate sgRNA.

0007The potential of the dCas9 complex for genome engineering purposes is immense. Its unique ability to bring proteins to specific sites in a genome programmed by the sgRNA in theory can be developed into a variety of site-specific genome engineering tools beyond nucleases, including deaminases (e.g., cytidine deamianses), transcriptional activators, transcriptional repressors, histone-modifying proteins, integrases, and recombinases.¹¹Some of these potential applications have recently been implemented through dCas9 fusions with transcriptional activators to afford RNA-guided transcriptional activators,^17,18transcriptional repressors, 16,19,20 and chromatin modification enzymes.²¹Simple co-expression of these fusions with a variety of sgRNAs results in specific expression of the target genes. These seminal studies have paved the way for the design and construction of readily programmable sequence-specific effectors for the precise manipulation of genomes.

0008Some aspects of the disclosure are based on the recognition that certain configurations of a nucleic acid programmable DNA binding protein (napDNAbp), for example CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein, and a cytidine deaminase domain fused by a linker are useful for efficiently deaminating target cytidine residues. Other aspects of this disclosure relate to the recognition that a nucleobase editing fusion protein with a cytidine deaminase domain fused to the N-terminus of a napDNAbp via a linker was capable of efficiently deaminating target nucleic acids in a double stranded DNA target molecule. See, for example, Examples 3 and 4 below, which demonstrate that the fusion proteins, which are also referred to herein as base editors, generate less indels and more efficiently deaminate target nucleic acids than other base editors, such as base editors without a UGI domain. Other aspects of this disclosure relate to the recognition that a nucleobase editing fusion protein with a cytidine deaminase domain fused to the N-terminus of napDNAbp via a linker perform base editing with higher efficiency and greatly improved product purity when the fusion protein is comprised of more than one UGI domain. See, for example, Example 17, which demonstrates that a fusion protein (e.g., base editor) comprising two UGI domains generates less indels and more efficiently deaminates target nucleic acids than other base editors, such as those comprising one UGI domain.

0009In some embodiments, the fusion protein comprises: (i) a nucleic acid programmable DNA binding protein (napDNAbp); (ii) a cytidine deaminase domain; and (iii) a uracil glycosylase inhibitor (UGI) domain, where the napDNAbp is a CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein. In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a CasX protein. In some embodiments, the CasX protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29 or 30. In some embodiments, the CasX protein comprises the amino acid sequence of SEQ ID NO: 29 or 30.

0010In some embodiments, the fusion protein comprises: (i) a nucleic acid programmable DNA binding protein (napDNAbp); (ii) a cytidine deaminase domain; (iii) a first uracil glycosylase inhibitor (UGI) domain; and (iv) a second uracil glycosylase inhibitor (UGI) domain, wherein the napDNAbp is a Cas9, dCas9, or Cas9 nickase protein. In some embodiments, the napDNAbp is a dCas9 protein. In some embodiments, the napDNAbp is a CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein. In some embodiments, the dCas9 protein is a <i>S. pyogenes </i>dCas9 (SpCas9d). In some embodiments, the dCas9 protein is a <i>S. pyogenes </i>dCas9 harboring a D10A mutation. In some embodiments, the dCas9 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 6 or 7. In some embodiments, the dCas9 protein comprises the amino acid sequence of SEQ ID NO: 6 or 7. In some embodiments, the dCas9 protein is a <i>S. aureus </i>dCas9 (SaCas9d). In some embodiments, the dCas9 protein is a <i>S. aureus </i>dCas9 harboring a D10A mutation. In some embodiments, the dCas9 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 33-36. In some embodiments, the dCas9 protein comprises the amino acid sequence of SEQ ID NO: 33-36.

0011In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a CasY protein. In some embodiments, the CasY protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 31. In some embodiments, the CasY protein comprises the amino acid sequence of SEQ ID NO: 31.

0012In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a Cpf1 or Cpf1 mutant protein. In some embodiments, the Cpf1 or Cpf1 mutant protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 9-24. In some embodiments, the Cpf1 or Cpf1 mutant protein comprises the amino acid sequence of any one of SEQ ID NOs: 9-24.

0013In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a C2c1 protein. In some embodiments, the C2c1 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 26. In some embodiments, the C2c1 protein comprises the amino acid sequence of SEQ ID NO: 26.

0014In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a C2c2 protein. In some embodiments, the C2c2 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 27. In some embodiments, the C2c2 protein comprises the amino acid sequence of SEQ ID NO: 27.

0015In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a C2c3 protein. In some embodiments, the C2c3 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 28. In some embodiments, the C2c3 protein comprises the amino acid sequence of SEQ ID NO: 28.

0016In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is an Argonaute protein. In some embodiments, the Argonaute protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 25. In some embodiments, the Argonaute protein comprises the amino acid sequence of SEQ ID NO: 25.

0017Some aspects of the disclosure are based on the recognition that fusion proteins provided herein are capable of generating one or more mutations (e.g., a C to T mutation) without generating a large proportion of indels. In some embodiments, any of the fusion proteins (e.g., base editing proteins) provided herein generate less than 10% indels. In some embodiments, any of the fusion proteins (e.g., base editing proteins) provided herein generate less than 10%, 9%, 8%, 7%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% indels.

0018In some embodiments, the fusion protein comprises a napDNAbp and an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, where the deaminase domain is fused to the N-terminus of the napDNAbp domain via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604). In some embodiments, the napDNAbp comprises the amino acid sequence of any of the napDNAbp provided herein. In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 76). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 74). In some embodiments, the deaminase is pmCDA1 (SEQ ID NO: 81). In some embodiments, the deaminase is human APOBEC3G (SEQ ID NO: 60). In some embodiments, the deaminase is a human APOBEC3G variant of any one of (SEQ ID NOs: 82-84). In some embodiments, the fusion protein comprises a napDNAbp and an apolipoprotein B mRNA-editing complex 1 catalytic polypeptide-like 3G (APOBEC3G) deaminase domain, wherein the deaminase domain is fused to the N-terminus of the napDNAbp domain via a linker of any length or composition (e.g., an amino acid sequence, a peptide, a polymer, or a bond). In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605).

0019In some embodiments, the fusion protein comprises a napDNAbp and a cytidine deaminase 1 (CDA1) deaminase domain, wherein the deaminase domain is fused to the N-terminus of the napDNAbp domain via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605). In some embodiments, the napDNAbp comprises the amino acid sequence of any of the napDNAbps provided herein.

0020In some embodiments, the fusion protein comprises a napDNAbp and an activation-induced cytidine deaminase (AID) deaminase domain, where the deaminase domain is fused to the N-terminus of the napDNAbp domain via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605). In some embodiments, the napDNAbp comprises the amino acid sequence of any of the napDNAbps provided herein.

0021Some aspects of the disclosure are based on the recognition that certain configurations of a napDNAbp, and a cytidine deaminase domain fused by a linker are useful for efficiently deaminating target cytidine residues. Other aspects of this disclosure relate to the recognition that a nucleobase editing fusion protein with an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain fused to the N-terminus of a napDNAbp via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604) was capable of efficiently deaminating target nucleic acids in a double stranded DNA target molecule. In some embodiments, the fusion protein comprises a napDNAbp domain and an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, where the deaminase domain is fused to the N-terminus of the napDNAbp via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604).

0022Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within a subject's genome, e.g., a human's genome. In some embodiments, fusion proteins of napDNAbp (e.g., CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein) and deaminases or deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of napDNAbp and deaminases or deaminase domains, are provided.

0023Some aspects of this disclosure provide fusion proteins comprising a napDNAbp as provided herein that is fused to a second protein (e.g., an enzymatic domain such as a cytidine deaminase domain), thus forming a fusion protein. In some embodiments, the second protein comprises an enzymatic domain, or a binding domain. In some embodiments, the enzymatic domain is a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain. In some embodiments, the enzymatic domain is a nucleic acid editing domain. In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytosine deaminase or a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase. In some embodiments, the deaminase is an APOBEC2 deaminase. In some embodiments, the deaminase is an APOBEC3 deaminase. In some embodiments, the deaminase is an APOBEC3A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase. In some embodiments, the deaminase is an APOBEC3E deaminase. In some embodiments, the deaminase is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). It should be appreciated that the deaminase may be from any suitable organism (e.g., a human or a rat). In some embodiments, the deaminase is from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 76). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 74). In some embodiments, the deaminase is pmCDA1.

0024Some aspects of this disclosure provide fusion proteins comprising: (i) a CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein domain comprising the amino acid sequence of SEQ ID NO: 32; and (ii) an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, wherein the deaminase domain is fused to the N-terminus of the napDNAbp via a linker comprising the amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 604). In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 76). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 74). In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 591. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 5737. In some embodiments, the deaminase is pmCDA1 (SEQ ID NO: 81). In some embodiments, the deaminase is human APOBEC3G (SEQ ID NO: 60). In some embodiments, the deaminase is a human APOBEC3G variant of any one of SEQ ID NOs: 82-84.

0025Other aspects of this disclosure relate to the recognition that fusion proteins comprising a deaminase domain, a napDNAbp domain and a uracil glycosylase inhibitor (UGI) domain demonstrate improved efficiency for deaminating target nucleotides in a nucleic acid molecule. Without wishing to be bound by any particular theory, cellular DNA-repair response to the presence of U:G heteroduplex DNA may be responsible for a decrease in nucleobase editing efficiency in cells. Uracil DNA glycosylase (UDG) catalyzes removal of U from DNA in cells, which may initiate base excision repair, with reversion of the U:G pair to a C:G pair as the most common outcome. As demonstrated herein, Uracil DNA Glycosylase Inhibitor (UGI) may inhibit human UDG activity. Without wishing to be bound by any particular theory, base excision repair may be inhibited by molecules that bind the single strand, block the edited base, inhibit UGI, inhibit base excision repair, protect the edited base, and/or promote “fixing” of the non-edited strand, etc. Thus, this disclosure contemplates fusion proteins comprising a napDNAbp-cytidine deaminase domain that is fused to a UGI domain.

0026Further aspects of this disclosure relate to the recognition that fusion proteins comprising a deaminase domain, a napDNAbp domain, and more than one uracil glycosylase inhibitor (UGI) domain (e.g., one, two, three, four, five, or more UGI domains) demonstrate improved efficiency for deaminating target nucleotides in a nucleic acid molecule and/or improved nucleic acid product purity. Without wishing to be bound by any particular theory, the addition of a second UGI domain may substantially decrease the access of UDG to the G:U base editing intermediate, thereby improving the efficiency of the base editing.

0027Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of modifying a specific nucleotide base without generating a significant proportion of indels. An “indel”, as used herein, refers to the insertion or deletion of a nucleotide base within a nucleic acid. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. In some embodiments, it is desirable to generate base editors that efficiently modify (e.g. mutate or deaminate) a specific nucleotide within a nucleic acid, without generating insertions or deletions (i.e., indels) in the nucleic acid. In certain embodiments, any of the base editors provided herein are capable of generating a greater proportion of intended modifications (e.g., point mutations or deaminations) versus indels.

0028In certain embodiments, any of the base editors provided herein are capable of generating a certain percentage of desired mutations. In some embodiments, the desired mutation is a C to T mutation. In some embodiments, the desired mutation is a C to A mutation, In some embodiments, the desired mutation is a C to G mutation. In some embodiments, any of the base editors provided herein are capable of generating at least 1% of desired mutations. In some embodiments, any of the base editors provided herein are capable of generating at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of desired mutations.

0029Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of efficiently generating an intended mutation, such as a point mutation, in a nucleic acid (e.g. a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations.

0030In some embodiments, the deaminase domain of the fusion protein is fused to the N-terminus of the napDNAbp domain. In some embodiments, the UGI domain is fused to the C-terminus of the napDNAbp domain. In some embodiments, the napDNAbp and the nucleic acid editing domain are fused via a linker. In some embodiments, the napDNAbp domain and the UGI domain are fused via a linker. In some embodiments, a second UGI domain is fused to the C-terminus of a first UGI domain. In some embodiments, the first UGI domain and the second UGI domain are fused via a linker.

0031In certain embodiments, linkers may be used to link any of the peptides or peptide domains of the invention. The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polpeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may included functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

0032In some embodiments, the linker comprises the amino acid sequence (GGGGS)_n(SEQ ID NO: 607), (G)_n(SEQ ID NO: 608), (EAAAK)_n(SEQ ID NO: 609), (GGS)_n(SEQ ID NO:610), (SGGS)_n(SEQ ID NO: 606), SGSETPGTSESATPES (SEQ ID NO: 604), (XP)_n(SEQ ID NO: 611), SGGS(GGS)_n(SEQ ID NO: 612), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605), or any combination thereof, wherein n is independently an integer between 1 and 30, and X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS)_n(SEQ ID NO: 610), wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGGS(GGS)_n(SEQ ID NO: 612), wherein n is 2. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605).

0033In some embodiments, the fusion protein comprises the structure [nucleic acid editing domain]-[optional linker sequence]-[napDNAbp]-[optional linker sequence]-[UGI]. In some embodiments, the fusion protein comprises the structure [nucleic acid editing domain]-[optional linker sequence]-[UGI]-[optional linker sequence]-[napDNAbp]; [UGI]-[optional linker sequence]-[nucleic acid editing domain]-[optional linker sequence]-[napDNAbp]; [UGI]-[optional linker sequence]-[napDNAbp]-[optional linker sequence]-[nucleic acid editing domain]; [napDNAbp]-[optional linker sequence]-[UGI]-[optional linker sequence]-[nucleic acid editing domain]; [napDNAbp]-[optional linker sequence]-[nucleic acid editing domain]-[optional linker sequence]-[UGI]; or [nucleic acid editing domain]-[optional linker sequence]-[napDNAbp]-[optional linker sequence]-[first UGI]-[optional linker sequence]-[second UGI].

0034In some embodiments, the nucleic acid editing domain comprises a deaminase. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase, or an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a cytidine deaminase 1 (CDA1). In some embodiments, the deaminase is a Lamprey CDA1 (pmCDA1) deaminase.

0035In some embodiments, the deaminase is from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is from a human. In some embodiments the deaminase is from a rat. In some embodiments, the deaminase is a rat APOBEC1 deaminase comprising the amino acid sequence set forth in (SEQ ID NO: 76). In some embodiments, the deaminase is a human APOBEC1 deaminase comprising the amino acid sequence set forth in (SEQ ID NO: 74). In some embodiments, the deaminase is pmCDA1 (SEQ ID NO: 81). In some embodiments, the deaminase is human APOBEC3G (SEQ ID NO: 60). In some embodiments, the deaminase is a human APOBEC3G variant of any one of (SEQ ID NOs: 82-84). In some embodiments, the deaminase is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 49-84.

0036In some embodiments, the UGI domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 134. In some embodiments, the UGI domain comprises the amino acid sequence as set forth in SEQ ID NO: 134.

0037Some aspects of this disclosure provide complexes comprising a napDNAbp fusion protein as provided herein, and a guide RNA bound to the napDNAbp.

0038Some aspects of this disclosure provide methods of using the napDNAbp, fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule (a) with a napDNAbp or a fusion protein as provided herein and with a guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence; or (b) with a napDNAbp, a napDNAbp fusion protein, or a napDNAbp or napDNAbp complex with a gRNA as provided herein.

0039Some aspects of this disclosure provide kits comprising a nucleic acid construct, comprising (a) a nucleotide sequence encoding a napDNAbp or a napDNAbp fusion protein as provided herein; and (b) a heterologous promoter that drives expression of the sequence of (a). In some embodiments, the kit further comprises an expression construct encoding a guide RNA backbone, wherein the construct comprises a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to a target sequence into the guide RNA backbone.

0040Some aspects of this disclosure provide polynucleotides encoding a napDNAbp of a fusion protein as provided herein. Some aspects of this disclosure provide vectors comprising such polynucleotides. In some embodiments, the vector comprises a heterologous promoter driving expression of polynucleotide.

0041Some aspects of this disclosure provide cells comprising a napDNAbp protein, a fusion protein, a nucleic acid molecule, and/or a vector as provided herein.

0042It should be appreciated that any of the fusion proteins provided herein that include a Cas9 domain (e.g. Cas9, nCas9, or dCas9) may be replaced with any of the napDNAbp provided herein, for example CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein.

0043The description of exemplary embodiments of the reporter systems above is provided for illustration purposes only and not meant to be limiting. Additional reporter systems, e.g., variations of the exemplary systems described in detail above, are also embraced by this disclosure.

0044The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

0045<figref idref="DRAWINGS">FIG. 1</figref> shows the deaminase activity of deaminases on single stranded DNA substrates. Single stranded DNA substrates using randomized PAM sequences (NNN PAM) were used as negative controls. Canonical PAM sequences used include the (NGG PAM).

0046<figref idref="DRAWINGS">FIG. 2</figref> shows the activity of Cas9:deaminase fusion proteins on single stranded DNA substrates.

0047<figref idref="DRAWINGS">FIG. 3</figref> illustrates double stranded DNA substrate binding by Cas9:deaminase:sgRNA complexes.

0048<figref idref="DRAWINGS">FIG. 4</figref> illustrates a double stranded DNA deamination assay.

0049<figref idref="DRAWINGS">FIG. 5</figref> demonstrates that Cas9 fusions can target positions 3-11 of double-stranded DNA target sequences (numbered according to the schematic in <figref idref="DRAWINGS">FIG. 5</figref>). Upper Gel: 1 μM rAPOBEC1-GGS-dCas9, 125 nM dsDNA, 1 equivalent sgRNA. Mid Gel: 1 μM rAPOBEC1-(GGS)₃(SEQ ID NO: 610)-dCas9, 125 nM dsDNA, 1 equivalent sgRNA. Lower Gel: 1.85 μM rAPOBEC1-XTEN-dCas9, 125 nM dsDNA, 1 equivalent sgRNA.

0050<figref idref="DRAWINGS">FIG. 6</figref> demonstrates that the correct guide RNA, e.g., the correct sgRNA, is required for deaminase activity.

0051<figref idref="DRAWINGS">FIG. 7</figref> illustrates the mechanism of target DNA binding of in vivo target sequences by deaminase-dCas9:sgRNA complexes.

0052<figref idref="DRAWINGS">FIG. 8</figref> shows successful deamination of exemplary disease-associated target sequences.

0053<figref idref="DRAWINGS">FIG. 9</figref> shows in vitro C→T editing efficiencies using His6-rAPOBEC1-XTEN-dCas9.

0054<figref idref="DRAWINGS">FIG. 10</figref> shows C→T editing efficiencies in HEK293T cells is greatly enhanced by fusion with UGI.

0055<figref idref="DRAWINGS">FIGS. 11A to 11C</figref> show NBE1 mediates specific, guide RNA-programmed C to U conversion in vitro. <figref idref="DRAWINGS">FIG. 11A</figref>: Nucleobase editing strategy. DNA with a target C at a locus specified by a guide RNA is bound by dCas9, which mediates the local denaturation of the DNA substrate. Cytidine deamination by a tethered APOBEC1 enzyme converts the target C to U. The resulting G:U heteroduplex can be permanently converted to an A:T base pair following DNA replication or repair. If the U is in the template DNA strand, it will also result in an RNA transcript containing a G to A mutation following transcription. <figref idref="DRAWINGS">FIG. 11B</figref>: Deamination assay showing an activity window of approximately five nucleotides. Following incubation of NBE1-sgRNA complexes with dsDNA substrates at 37° C. for 2 h, the 5′ fluorophore-labeled DNA was isolated and incubated with USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37° C. for 1 h to induce DNA cleavage at the site of any uracils. The resulting DNA was resolved on a denaturing polyacrylamide gel, and any fluorophore-linked strands were visualized. Each lane is labeled according to the position of the target C within the protospacer, or with “-” if no target C is present, counting the base distal from the PAM as position 1. <figref idref="DRAWINGS">FIG. 11C</figref>: Deaminase assay showing the sequence specificity and sgRNA-dependence of NBE1. The DNA substrate with a target C at position 7 was incubated with NBE1 as in <figref idref="DRAWINGS">FIG. 11B</figref> with either the correct sgRNA, a mismatched sgRNA, or no sgRNA. No C to U editing is observed with the mismatched sgRNA or with no sgRNA. The positive control sample contains a DNA sequence with a U synthetically incorporated at position 7.

0056<figref idref="DRAWINGS">FIGS. 12A to 12B</figref> show effects of sequence context and target C position on nucleobase editing efficiency in vitro. <figref idref="DRAWINGS">FIG. 12A</figref>: Effect of changing the sequence surrounding the target C on editing efficiency in vitro. The deamination yield of 80% of targeted strands (40% of total sequencing reads from both strands) for C₇in the protospacer sequence 5′-TTATTTCGTGGATTTATTTA-3′(SEQ ID NO: 591) was defined as 1.0, and the relative deamination efficiencies of substrates containing all possible single-base mutations at positions 1-6 and 8-13 are shown. Values and error bars reflect the mean and standard deviation of two or more independent biological replicates performed on different days. <figref idref="DRAWINGS">FIG. 12B</figref>: Positional effect of each NC motif on editing efficiency in vitro. Each NC target motif was varied from positions 1 to 8 within the protospacer as indicated in the sequences shown on the right (the PAM shown in red, the protospacer plus one base 5′ to the protospacer are also shown). The percentage of total sequence reads containing T at each of the numbered target C positions following incubation with NBE1 is shown in the graph. Note that the maximum possible deamination yield in vitro is 50% of total sequencing reads (100% of targeted strands). Values and error bars reflect the mean and standard deviation of two or three independent biological replicates performed on different days. <figref idref="DRAWINGS">FIG. 12B</figref> depicts SEQ ID NOs: 619 through 626 from top to bottom, respectively.

0057<figref idref="DRAWINGS">FIGS. 13A to 13C</figref> show nucleobase editing in human cells. <figref idref="DRAWINGS">FIG. 13A</figref>: Protospacer and PAM sequences of the six mammalian cell genomic loci targeted by nucleobase editors. Target Cs are indicated with subscripted numbers corresponding to their positions within the protospacer. <figref idref="DRAWINGS">FIG. 13A</figref> depicts SEQ ID NOs: 127 through 132 from top to bottom, respectively. <figref idref="DRAWINGS">FIG. 13B</figref>: HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and an appropriate sgRNA. Three days after transfection, genomic DNA was extracted and analyzed by high-throughput DNA sequencing at the six loci. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown for NBE1, NBE2, and NBE3 at all six genomic loci, and for wt Cas9 with a donor HDR template at three of the six sites (EMX1, HEK293 site 3, and HEK293 site 4). Values and error bars reflect the mean and standard deviation of three independent biological replicates performed on different days. <figref idref="DRAWINGS">FIG. 13C</figref>: Frequency of indel formation, calculated as described in the Methods, is shown following treatment of HEK293T cells with NBE2 and NBE3 for all six genomic loci, or with wt Cas9 and a single-stranded DNA template for HDR at three of the six sites (EMX1, HEK293 site 3, and HEK293 site 4). Values reflect the mean of at least three independent biological replicates performed on different days.

0058<figref idref="DRAWINGS">FIGS. 14A to 14C</figref> show NBE2- and NBE3-mediated correction of three disease-relevant mutations in mammalian cells. For each site, the sequence of the protospacer is indicated to the right of the name of the mutation, with the PAM and the base responsible for the mutation indicated in bold with a subscripted number corresponding to its position within the protospacer. The amino acid sequence above each disease-associated allele is shown, together with the corrected amino acid sequence following nucleobase editing in red. Underneath each sequence are the percentages of total sequencing reads with the corresponding base. Cells were nucleofected with plasmids encoding NBE2 or NBE3 and an appropriate sgRNA. Two days after nucleofection, genomic DNA was extracted and analyzed by HTS to assess pathogenic mutation correction. <figref idref="DRAWINGS">FIG. 14A</figref>: The Alzheimer's disease-associated APOE4 allele is converted to APOE3′ in mouse astrocytes by NBE3 in 11% of total reads (44% of nucleofected astrocytes). Two nearby Cs are also converted to Ts, but with no change to the predicted sequence of the resulting protein (SEQ ID NO: 627). <figref idref="DRAWINGS">FIG. 14B</figref> The cancer-associated p53 N239D mutation is corrected by NBE2 in 11% of treated human lymphoma cells (12% of nucleofected cells) that are heterozygous for the mutation (SEQ ID NO: 628). <figref idref="DRAWINGS">FIG. 14C</figref> The p53 Y163C mutation is corrected by NBE3 in 7.6% of nucleofected human breast cancer cells (SEQ ID NO: 629).

0059<figref idref="DRAWINGS">FIGS. 15A to 15D</figref> show effects of deaminase-dCas9 linker length and composition on nucleobase editing. Gel-based deaminase assay showing the deamination window of nucleobase editors with deaminase-Cas9 linkers of GGS (<figref idref="DRAWINGS">FIG. 15A</figref>), (GGS)₃(SEQ ID NO: 610) (<figref idref="DRAWINGS">FIG. 15B</figref>), XTEN (<figref idref="DRAWINGS">FIG. 15C</figref>), or (GGS)₇(SEQ ID NO: 610) (<figref idref="DRAWINGS">FIG. 15D</figref>). Following incubation of 1.85 M editor-sgRNA complexes with 125 nM dsDNA substrates at 37° C. for 2 h, the dye-conjugated DNA was isolated and incubated with USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37° C. for an additional hour to cleave the DNA backbone at the site of any uracils. The resulting DNA was resolved on a denaturing polyacrylamide gel, and the dye-conjugated strand was imaged. Each lane is numbered according to the position of the target C within the protospacer, or with - if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8.

0060<figref idref="DRAWINGS">FIGS. 16A to 16B</figref> show NBE1 is capable of correcting disease-relevant mutations in vitro. <figref idref="DRAWINGS">FIG. 16A</figref>: Protospacer and PAM sequences of seven disease-relevant mutations. The disease-associated target C in each case is indicated with a subscripted number reflecting its position within the protospacer. For all mutations except both APOE4 SNPs, the target C resides in the template (non-coding) strand. <figref idref="DRAWINGS">FIG. 16A</figref> depicts SEQ ID NOs: 631 through 636 from top to bottom, respectively. <figref idref="DRAWINGS">FIG. 16B</figref>: Deaminase assay showing each dsDNA oligonucleotide before (−) and after (+) incubation with NBE1, DNA isolation, and incubation with USER enzymes to cleave DNA at positions containing U. Positive control lanes from incubation of synthetic oligonucleotides containing U at various positions within the protospacer with USER enzymes are shown with the corresponding number indicating the position of the U.

0061<figref idref="DRAWINGS">FIG. 17</figref> shows processivity of NBE1. The protospacer and PAM of a 60-mer DNA oligonucleotide containing eight consecutive Cs is shown at the top. The oligonucleotide (125 nM) was incubated with NBE1 (2 μM) for 2 h at 37° C. The DNA was isolated and analyzed by high-throughput sequencing. Shown are the percent of total reads for the most frequent nine sequences observed. The vast majority of edited strands (&gt;93%) have more than one C converted to T. This figure depicts SEQ ID NO: 309.

0062<figref idref="DRAWINGS">FIGS. 18A to 18H</figref> show the effect of fusing UGI to NBE1 to generate NBE2. <figref idref="DRAWINGS">FIG. 18A</figref>: Protospacer and PAM sequences of the six mammalian cell genomic loci targeted with nucleobase editors. Editable Cs are indicated with labels corresponding to their positions within the protospacer. <figref idref="DRAWINGS">FIG. 18A</figref> depicts SEQ ID NOs: 127 through 132 from top to bottom, respectively. <figref idref="DRAWINGS">FIGS. 18B to 18G</figref>: HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE1 and UGI, and an appropriate sgRNA. Three days after transfection, genomic DNA was extracted and analyzed by high-throughput DNA sequencing at the six loci. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown for NBE1, NBE1 and UGI, and NBE2 at all six genomic loci. <figref idref="DRAWINGS">FIG. 18H</figref>: C to T mutation rates at 510 Cs surrounding the protospacers of interest for NBE1, NBE1 plus UGI on a separate plasmid, NBE2, and untreated cells are shown. The data show the results of 3,000,000 DNA sequencing reads from 1.5×106 cells. Values reflect the mean of at least two biological experiments conducted on different days.

0063<b>19</b> shows nucleobase editing efficiencies of NBE2 in U2OS and HEK293T cells. Cellular C to T conversion percentages by NBE2 are shown for each of the six targeted genomic loci in HEK293T cells and U2OS cells. HEK293T cells were transfected using LIPOFECTAMINE® 2000 transfection reagent, and U2OS cells were nucleofected. U2OS nucleofection efficiency was 74%. Three days after plasmid delivery, genomic DNA was extracted and analyzed for nucleobase editing at the six genomic loci by HTS. Values and error bars reflect the mean and standard deviation of at least two biological experiments done on different days.

0064<figref idref="DRAWINGS">FIG. 20</figref> shows nucleobase editing persists over multiple cell divisions. Cellular C to T conversion percentages by NBE2 are displayed at two genomic loci in HEK293T cells before and after passaging the cells. HEK293T cells were transfected using LIPOFECTAMINE® 2000 transfection reagent. Three days post transfection, the cells were harvested and split in half. One half was subjected to HTS analysis, and the other half was allowed to propagate for approximately five cell divisions, then harvested and subjected to HTS analysis.

0065<figref idref="DRAWINGS">FIG. 21</figref> shows genetic variants from ClinVar that can be corrected in principle by nucleobase editing. The NCBI ClinVar database of human genetic variations and their corresponding phenotypes⁶⁸was searched for genetic diseases that can be corrected by current nucleobase editing technologies. The results were filtered by imposing the successive restrictions listed on the left. The x-axis shows the number of occurrences satisfying that restriction and all above restrictions on a logarithmic scale.

0066<figref idref="DRAWINGS">FIGS. 22A to 22B</figref> shows in vitro identification of editable Cs in six genomic loci. Synthetic 80-mers with sequences matching six different genonic sites were incubated with NBE1 then analyzed for nucleobase editing via HTS. For each site, the sequence of the protospacer is indicated to the right of the name of the site, with the PAM highlighted in red. Underneath each sequence are the percentages of total DNA sequencing reads with the corresponding base. A target C was considered as “editable” if the in vitro conversion efficiency is &gt;10%. Note that maximum yields are 50% of total DNA sequencing reads since the non-targeted strand is not a substrate for nucleobase editing. This figure depicts SEQ ID NOs: 127 through 132 from top to bottom, respectively.

0067<figref idref="DRAWINGS">FIGS. 23A to 2C</figref> shows activities of NBE1, NBE2, and NBE3 at EMX1 off-targets. HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and a sgRNA matching the EMX1 sequence using LIPOFECTAMINE® 2000 transfection reagent. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus the top ten known Cas9 off-target loci for the EMX1 sgRNA, as previously determined using the GUIDE-seq method⁵⁵. EMX1 off-target 5 locus did not amplify and is not shown. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for NBE1, NBE2, and NBE3. On the far right are displayed the total number of sequencing reads reported for each sequence. This figure depicts SEQ ID NOs: 127, and 637 through 645 from top to bottom, respectively.

0068<figref idref="DRAWINGS">FIGS. 24A to 24B</figref> shows activities of NBE1, NBE2, and NBE3 at FANCF off-targets. HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and a sgRNA matching the FANCF sequence using LIPOFECTAMINE® 2000 transfection reagent. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus all of the known Cas9 off-target loci for the FANCF sgRNA, as previously determined using the GUIDE-seq method⁵⁵. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for NBE1, NBE2, and NBE3. On the far right are displayed the total number of sequencing reads reported for each sequence. This figure depicts SEQ ID NOs: 128 and 646 through 653 from top to bottom, respectively.

0069<figref idref="DRAWINGS">FIG. 25</figref> shows activities of NBE1, NBE2, and NBE3 at HEK293 site 2 off-targets. HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and a sgRNA matching the HEK293 site 2 sequence using LIPOFECTAMINE® 2000 transfection reagent. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus all of the known Cas9 off-target loci for the HEK293 site 2 sgRNA, as previously determined using the GUIDE-seq method⁵⁵. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for NBE1, NBE2, and NBE3. On the far right are displayed the total number of sequencing reads reported for each sequence. This figure depicts SEQ ID NOs: 129, 654, and 655 from top to bottom, respectively.

0070<figref idref="DRAWINGS">FIG. 26</figref> shows activities of NBE1, NBE2, and NBE3 at HEK293 site 3 off-targets. HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and a sgRNA matching the HEK293 site 3 sequence using LIPOFECTAMINE® 2000 transfection reagent. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus all of the known Cas9 off-target loci for the HEK293 site 3 sgRNA, as previously determined using the GUIDE-seq method.⁵⁵Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for NBE1, NBE2, and NBE3. On the far right are displayed the total number of sequencing reads reported for each sequence. This figure depicts SEQ ID NOs: 130 and 656 through 660 from top to bottom, respectively.

0071<figref idref="DRAWINGS">FIGS. 27A to 27B</figref> shows activities of NBE1, NBE2, and NBE3 at HEK293 site 4 off-targets. HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and a sgRNA matching the HEK293 site 4 sequence using LIPOFECTAMINE® 2000 transfection reagent. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus the top ten known Cas9 off-target loci for the HEK293 site 4 sgRNA, as previously determined using the GUIDE-seq method.⁵⁵Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for NBE1, NBE2, and NBE3. On the far right are displayed the total number of sequencing reads reported for each sequence. This figure depicts SEQ ID NOs: 131 and 661 through 670 from top to bottom, respectively.

0072<figref idref="DRAWINGS">FIG. 28</figref> shows non-target C mutation rates. Shown here are the C to T mutation rates at 2,500 distinct cytosines surrounding the six on-target and 34 off-target loci tested, representing a total of 14,700,000 sequence reads derived from approximately 1.8×106 cells.

0073<figref idref="DRAWINGS">FIGS. 29A to 29C</figref> show base editing in human cells. <figref idref="DRAWINGS">FIG. 29A</figref> shows possible base editing outcomes in mammalian cells. Initial editing resulted in a U:G mismatch. Recognition and excision of the U by uracil DNA glycosylase (UDG) initiated base excision repair (BER), which lead to reversion to the C:G starting state. BER was impeded by BE2 and BE3, which inhibited UDG. The U:G mismatch was also processed by mismatch repair (MMR), which preferentially repaired the nicked strand of a mismatch. BE3 nicked the non-edited strand containing the G, favoring resolution of the U:G mismatch to the desired U:A or T:A outcome. <figref idref="DRAWINGS">FIG. 29B</figref> shows HEK293T cells treated as described in the Materials and Methods in the Examples below. The percentage of total DNA sequencing read with Ts at the target positions indicated show treatment with BE1, BE2, or BE3, or for treatment with wt Cas9 with a donor HDR template. <figref idref="DRAWINGS">FIG. 29C</figref> shows frequency of indel formation following the treatment in <figref idref="DRAWINGS">FIG. 29B</figref>. Values are listed in <figref idref="DRAWINGS">FIG. 34</figref>. For <figref idref="DRAWINGS">FIGS. 29B and 29C</figref>, values and error bars reflect the mean and s.d. of three independent biological replicates performed on different days.

0074<figref idref="DRAWINGS">FIGS. 30A to 30B</figref> show BE3-mediated correction of two disease-relevant mutations in mammalian cells. The sequence of the protospacer is shown to the right of the mutation, with the PAM and the target base in red with a subscripted number indicating its position within the protospacer. Underneath each sequence are the percentages of total sequencing reads with the corresponding base. Cells were treated as described in the Materials and Methods. <figref idref="DRAWINGS">FIG. 30A</figref> shows the Alzheimer's disease-associated APOE4 allele converted to APOE3r in mouse astrocytes by BE3 in 74.9% of total reads. Two nearby Cs were also converted to Ts, but with no change to the predicted sequence of the resulting protein. Identical treatment of these cells with wt Cas9 and donor ssDNA results in only 0.3% correction, with 26.1% indel formation. This figure depicts SEQ ID NOs: 671 and 627. <figref idref="DRAWINGS">FIG. 30B</figref> shows the cancer associated p53 Y163C mutation corrected by BE3 in 7.6% of nucleofected human breast cancer cells with 0.7% indel formation. Identical treatment of these cells with wt Cas9 and donor ssDNA results in no mutation correction with 6.1% indel formation. This figure depicts SEQ ID NOs: 672 and 629.

0075<figref idref="DRAWINGS">FIGS. 31A to 31B</figref> shows activities of BE1, BE2, and BE3 at HEK293 site 2 off-targets. HEK293T cells were transfected with plasmids expressing BE1, BE2, or BE3 and a sgRNA matching the HEK293 site 2 sequence using LIPOFECTAMINE® 2000 transfection reagent. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus all of the known Cas9 and dCas9 off-target loci for the HEK293 site 2 sgRNA, as previously determined by Joung and coworkers using the GUIDE-seq method (63), and Adli and coworkers using chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) experiments (18). Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for BE1, BE2, and BE3. On the far right are displayed the total number of sequencing reads reported, and the ChIP-seq signal intensity reported for each sequence. This figure depicts SEQ ID NOs: 129, 654, 655 and 673 to 677 from top to bottom, respectively.

0076<figref idref="DRAWINGS">FIGS. 32A to 32B</figref> shows activities of BE1, BE2, and BE3 at HEK293 site 3 off-targets. HEK293T cells were transfected with plasmids expressing BE1, BE2, or BE3 and a sgRNA matching the HEK293 site 3 sequence using LIPOFECTAMINE® 2000 transfection reagent. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus all of the known Cas9 off-target loci and the top five known dCas9 off-target loci for the HEK293 site 3 sgRNA, as previously determined by Joung and coworkers using the GUIDE-seq method⁵⁴, and using chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) experiments⁶¹. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for BE1, BE2, and BE3. On the far right are displayed the total number of sequencing reads reported, and the ChIP-seq signal intensity reported for each sequence. This figure depicts SEQ ID NOs: 130, 656 to 660 and 678-682 from top to bottom, respectively.

0077<figref idref="DRAWINGS">FIGS. 33A to 33C</figref> shows activities of BE1, BE2, and BE3 at HEK293 site 4 off-targets. HEK293T cells were transfected with plasmids expressing BE1, BE2, or BE3 and a sgRNA matching the HEK293 site 4 sequence using LIPOFECTAMINE® 2000 transfection reagent. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus the top ten known Cas9 off-target loci and the top five known dCas9 off-target loci for the HEK293 site 4 sgRNA, as previously determined using the GUIDE-seq method⁵⁴, and using chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) experiments⁶¹. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for BE1, BE2, and BE3. On the far right are displayed the total number of sequencing reads reported, and the ChIP-seq signal intensity reported for each sequence. This figure depicts SEQ ID NOs: 131, 661 to 670, 683 and 684 from top to bottom, respectively.

0078<figref idref="DRAWINGS">FIGS. 34A to 34B</figref> shows mutation rates of non-protospacer bases following BE3-mediated correction of the Alzheimer's disease-associated APOE4 allele to APOE3r in mouse astrocytes. The DNA sequence of the 50 bases on either side of the protospacer from <figref idref="DRAWINGS">FIG. 30A</figref> and <figref idref="DRAWINGS">FIG. 34B</figref> is shown with each base's position relative to the protospacer. The side of the protospacer distal to the PAM is designated with positive numbers, while the side that includes the PAM is designated with negative numbers, with the PAM. Underneath each sequence are the percentages of total DNA sequencing reads with the corresponding base for untreated cells, for cells treated with BE3 and an sgRNA targeting the APOE4 C158R mutation, or for cells treated with BE3 and an sgRNA targeting the VEGFA locus. Neither BE3-treated sample resulted in mutation rates above those of untreated controls. This figure depicts SEQ ID NOs: 685 to 688 from top to bottom, respectively.

0079<figref idref="DRAWINGS">FIGS. 35A to 35B</figref> shows mutation rates of non-protospacer bases following BE3-mediated correction of the cancer-associated p53 Y163C mutation in HCC1954 human cells. The DNA sequence of the 50 bases on either side of the protospacer from <figref idref="DRAWINGS">FIG. 30B</figref> and <figref idref="DRAWINGS">FIG. 39B</figref> is shown with each base's position relative to the protospacer. The side of the protospacer distal to the PAM is designated with positive numbers, while the side that includes the PAM is designated with negative numbers, with the PAM. Underneath each sequence are the percentages of total sequencing reads with the corresponding base for untreated cells, for cells treated with BE3 and an sgRNA targeting the TP53 Y163C mutation, or for cells treated with BE3 and an sgRNA targeting the VEGFA locus. Neither BE3-treated sample resulted in mutational rates above those of untreated controls. This figure depicts SEQ ID NOs: 689 to 692 from top to bottom, respectively.

0080<figref idref="DRAWINGS">FIGS. 36A to 36F</figref> show the effects of deaminase, linker length, and linker composition on base editing. <figref idref="DRAWINGS">FIG. 36A</figref> shows a gel-based deaminase assay showing activity of rAPOBEC1, pmCDA1, hAID, hAPOBEC3G, rAPOBEC1-GGS-dCas9, rAPOBEC1-(GGS)₃(SEQ ID NO: 610)-dCas9, and dCas9-(GGS)₃(SEQ ID NO: 610)-rAPOBEC1 on ssDNA. Enzymes were expressed in a mammalian cell lysate-derived in vitro transcription-translation system and incubated with 1.8 μM dye-conjugated ssDNA and USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37° C. for 2 hours. The resulting DNA was resolved on a denaturing polyacrylamide gel and imaged. The positive control is a sequence with a U synthetically incorporated at the same position as the target C. <figref idref="DRAWINGS">FIG. 36B</figref> shows coomassie-stained denaturing PAGE gel of the expressed and purified proteins used in <figref idref="DRAWINGS">FIGS. 36C to 36F</figref>. <figref idref="DRAWINGS">FIGS. 36C to 36F</figref> show gel-based deaminase assay showing the deamination window of base editors with deaminase-Cas9 linkers of GGS (<figref idref="DRAWINGS">FIG. 36C</figref>), (GGS)₃(SEQ ID NO: 610) (<figref idref="DRAWINGS">FIG. 36D</figref>), XTEN (<figref idref="DRAWINGS">FIG. 36E</figref>), or (GGS)₇(SEQ ID NO: 610) (<figref idref="DRAWINGS">FIG. 36F</figref>). Following incubation of 1.85 μM deaminase-dCas9 fusions complexed with sgRNA with 125 nM dsDNA substrates at 37° C. for 2 hours, the dye-conjugated DNA was isolated and incubated with USER enzyme at 37° C. for 1 hour to cleave the DNA backbone at the site of any uracils. The resulting DNA was resolved on a denaturing polyacrylamide gel, and the dye-conjugated strand was imaged. Each lane is numbered according to the position of the target C within the protospacer, or with - if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8.

0081<figref idref="DRAWINGS">FIGS. 37A to 37C</figref> show BE1 base editing efficiencies are dramatically decreased in mammalian cells. <figref idref="DRAWINGS">FIG. 37A</figref> Protospacer and PAM sequences of the six mammalian cell genomic loci targeted by base editors. Target Cs are indicated in red with subscripted numbers corresponding to their positions within the protospacer. <figref idref="DRAWINGS">FIG. 37B</figref> shows synthetic 80-mers with sequences matching six different genomic sites were incubated with BE1 then analyzed for base editing by HTS. For each site, the sequence of the protospacer is indicated to the right of the name of the site, with the PAM. Underneath each sequence are the percentages of total DNA sequencing reads with the corresponding base. We considered a target C as “editable” if the in vitro conversion efficiency is &gt;10%. Note that maximum yields are 50% of total DNA sequencing reads since the non-targeted strand is unaffected by BE1. Values are shown from a single experiment. <figref idref="DRAWINGS">FIG. 37C</figref> shows HEK293T cells were transfected with plasmids expressing BE1 and an appropriate sgRNA. Three days after transfection, genomic DNA was extracted and analyzed by high-throughput DNA sequencing at the six loci. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown for BE1 at all six genomic loci. Values and error bars of all data from HEK293T cells reflect the mean and standard deviation of three independent biological replicates performed on different days. <figref idref="DRAWINGS">FIG. 37A</figref> depicts SEQ ID NOs: 127 to 132 from top to bottom, respectively. <figref idref="DRAWINGS">FIG. 37B</figref> depicts SEQ ID NOs: 127 to 132 from top to bottom, respectively.

0082<figref idref="DRAWINGS">FIG. 38</figref> shows base editing persists over multiple cell divisions. Cellular C to T conversion percentages by BE2 and BE3 are shown for HEK293 sites 3 and 4 in HEK293T cells before and after passaging the cells. HEK293T cells were nucleofected with plasmids expressing BE2 or BE3 and an sgRNA targeting HEK293 site 3 or 4. Three days after nucleofection, the cells were harvested and split in half. One half was subjected to HTS analysis, and the other half was allowed to propagate for approximately five cell divisions, then harvested and subjected to HTS analysis. Values and error bars reflect the mean and standard deviation of at least two biological experiments.

0083<figref idref="DRAWINGS">FIGS. 39A to 39C</figref> show non-target C/G mutation rates. Shown here are the C to T and G to A mutation rates at 2,500 distinct cytosines and guanines surrounding the six on-target and 34 off-target loci tested, representing a total of 14,700,000 sequence reads derived from approximately 1.8×10⁶cells. <figref idref="DRAWINGS">FIGS. 39A and 39B</figref> show cellular non-target C to T and G to A conversion percentages by BE1, BE2, and BE3 are plotted individually against their positions relative to a protospacer for all 2,500 cytosines/guanines. The side of the protospacer distal to the PAM is designated with positive numbers, while the side that includes the PAM is designated with negative numbers. <figref idref="DRAWINGS">FIG. 39C</figref> shows average non-target cellular C to T and G to A conversion percentages by BE1, BE2, and BE3 are shown, as well as the highest and lowest individual conversion percentages.

0084<figref idref="DRAWINGS">FIGS. 40A to 40B</figref> show additional data sets of BE3-mediated correction of two disease-relevant mutations in mammalian cells. For each site, the sequence of the protospacer is indicated to the right of the name of the mutation, with the PAM and the base responsible for the mutation indicated in red bold with a subscripted number corresponding to its position within the protospacer. The amino acid sequence above each disease-associated allele is shown, together with the corrected amino acid sequence following base editing. Underneath each sequence are the percentages of total sequencing reads with the corresponding base. Cells were nucleofected with plasmids encoding BE3 and an appropriate sgRNA. Two days after nucleofection, genomic DNA was extracted from the nucleofected cells and analyzed by HTS to assess pathogenic mutation correction. <figref idref="DRAWINGS">FIG. 40A</figref> shows the Alzheimer's disease-associated APOE4 allele is converted to APOE3r in mouse astrocytes by BE3 in 58.3% of total reads only when treated with the correct sgRNA. Two nearby Cs are also converted to Ts, but with no change to the predicted sequence of the resulting protein. Identical treatment of these cells with wt Cas9 and donor ssDNA results in 0.2% correction, with 26.7% indel formation. FIG. <b>40</b>B shows the cancer-associated p53 Y163C mutation is corrected by BE3 in 3.3% of nucleofected human breast cancer cells only when treated with the correct sgRNA. Identical treatment of these cells with wt Cas9 and donor ssDNA results in no detectable mutation correction with 8.0% indel formation. <figref idref="DRAWINGS">FIGS. 40A to 40B</figref> depict SEQ ID NOs: 671, 627, 672 and 629.

0085<figref idref="DRAWINGS">FIG. 41</figref> shows a schematic representation of an exemplary USER (Uracil-Specific Excision Reagent) Enzyme-based assay, which may be used to test the activity of various deaminases on single-stranded DNA (ssDNA) substrates.

0086<figref idref="DRAWINGS">FIG. 42</figref> is a schematic of the pmCDA-nCas9-UGI-NLS construct and its activity at the HeK-3 site relative to the base editor (rAPOBEC1) and the negative control (untreated). This figure depicts SEQ ID NO: 693.

0087<figref idref="DRAWINGS">FIG. 43</figref> is a schematic of the pmCDA1-XTEN-nCas9-UGI-NLS construct and its activity at the HeK-3 site relative to the base editor (rAPOBEC1) and the negative control (untreated). This figure depicts SEQ ID NO: 694.

0088<figref idref="DRAWINGS">FIG. 44</figref> shows the percent of total sequencing reads with target C converted to T using cytidine deaminases (CDA) or APOBEC.

0089<figref idref="DRAWINGS">FIG. 45</figref> shows the percent of total sequencing reads with target C converted to A using deaminases (CDA) or APOBEC.

0090<figref idref="DRAWINGS">FIG. 46</figref> shows the percent of total sequencing reads with target C converted to G using deaminases (CDA) or APOBEC.

0091<figref idref="DRAWINGS">FIG. 47</figref> is a schematic of the huAPOBEC3G-XTEN-nCas9-UGI-NLS construct and its activity at the HeK-2 site relative to a mutated form (huAPOBEC3G*(D316R_D317R)-XTEN-nCas9-UGI-NLS, the base editor (rAPOBEC1) and the negative control (untreated). This figure depicts SEQ ID NO: 695.

0092<figref idref="DRAWINGS">FIG. 48</figref> shows the schematic of the LacZ construct used in the selection assay of Example 7.

0093<figref idref="DRAWINGS">FIGS. 49A to 49B</figref> shows reversion data from different plasmids and constructs.

0094<figref idref="DRAWINGS">FIG. 50</figref> shows the verification of lacZ reversion and the purification of reverted clones.

0095<figref idref="DRAWINGS">FIG. 51</figref> is a schematic depicting a deamination selection plasmid used in Example 7.

0096<figref idref="DRAWINGS">FIG. 52</figref> shows the results of a chloramphenicol reversion assay (pmCDA1 fusion).

0097<figref idref="DRAWINGS">FIGS. 53A to 53B</figref> demonstrated DNA correction induction of two constructs.

0098<figref idref="DRAWINGS">FIG. 54</figref> shows the results of a chloramphenicol reversion assay (huAPOBEC3G fusion).

0099<figref idref="DRAWINGS">FIGS. 55A to 55B</figref> shows the activities of BE3 and HF-BE3 at EMX1 off-targets. The sequences, from top to bottom, correspond to SEQ ID NOs: 127 and 637-645.

0100<figref idref="DRAWINGS">FIG. 56</figref> shows on-target base editing efficiencies of BE3 and HF-BE3.

0101<figref idref="DRAWINGS">FIG. 57</figref> is a graph demonstrating that mutations affect cytidine deamination with varying degrees. Combinations of mutations that each slightly impairs catalysis allow selective deamination at one position over others. The FANCF site was GGAATC₆C₇C₈TTC₁₁TGCAGCACCTGG (SEQ ID NO: 128).

0102<figref idref="DRAWINGS">FIG. 58</figref> is a schematic depicting next generation base editors.

0103<figref idref="DRAWINGS">FIG. 59</figref> is a schematic illustrating new base editors made from Cas9 variants.

0104<figref idref="DRAWINGS">FIG. 60</figref> shows the base-edited percentage of different NGA PAM sites.

0105<figref idref="DRAWINGS">FIG. 61</figref> shows the base-edited percentage of cytidines using NGCG PAM EMX (VRER BE3) and the C₁TC₃C₄C₅ATC₈AC₁₀ATCAACCGGT (SEQ ID NO: 696) spacer.

0106<figref idref="DRAWINGS">FIG. 62</figref> shows the based-edited percentages resulting from different NNGRRT PAM sites.

0107<figref idref="DRAWINGS">FIG. 63</figref> shows the based-edited percentages resulting from different NNHRRT PAM sites.

0108<figref idref="DRAWINGS">FIGS. 64A to 64C</figref> show the base-edited percentages resulting from different TTTN PAM sites using Cpf1 BE2. The spacers used were: TTTCCTC₃C₄C₅C₆C₇C₈C₉AC₁₁AGGTAGAACAT (<figref idref="DRAWINGS">FIG. 64A</figref>, SEQ ID NO: 697), TTTCC₁C₂TC₄TGTC₈C₉AC₁₁ACCCTCATCCTG (<figref idref="DRAWINGS">FIG. 64B</figref>, SEQ ID NO: 698), and TTTCC₁C₂C₃AGTC₇C₈TC₁₀C₁₁AC₁₃AC₁₅C₁₆C₁₇TGAAAC (<figref idref="DRAWINGS">FIG. 64C</figref>, SEQ ID NO: 699).

0109<figref idref="DRAWINGS">FIG. 65</figref> is a schematic depicting selective deamination as achieved through kinetic modulation of cytidine deaminase point mutagenesis.

0110<figref idref="DRAWINGS">FIG. 66</figref> is a graph showing the effect of various mutations on the deamination window probed in cell culture with multiple cytidines in the spacer. The spacer used was: TGC₃C₄C₅C₆TC₈C₉C₁₀TC₁₂C₁₃C₁₄TGGCCC (SEQ ID NO: 700).

0111<figref idref="DRAWINGS">FIG. 67</figref> is a graph showing the effect of various mutations on the deamination window probed in cell culture with multiple cytidines in the spacer. The spacer used was: AGAGC₅C₆C₇C₈C₉C₁₀C₁₁TC₁₃AAAGAGA (SEQ ID NO: 701).

0112<figref idref="DRAWINGS">FIG. 68</figref> is a graph showing the effect of various mutations on the FANCF site with a limited number of cytidines. The spacer used was: GGAATC₆C₇C₈TTC₁₁TGCAGCACCTGG (SEQ ID NO: 128). Note that the triple mutant (W90Y, R126E, R132E) preferentially edits the cytidine at the sixth position.

0113<figref idref="DRAWINGS">FIG. 69</figref> is a graph showing the effect of various mutations on the HEK3 site with a limited number of cytidines. The spacer used was: GGCC₄C₅AGACTGAGCACGTGATGG (SEQ ID NO: 702). Note that the double and triple mutants preferentially edit the cytidine at the fifth position over the cytidine in the fourth position.

0114<figref idref="DRAWINGS">FIG. 70</figref> is a graph showing the effect of various mutations on the EMX1 site with a limited number of cytidines. The spacer used was: GAGTC₅C₆GAGCAGAAGAAGAAGGG (SEQ ID NO: 703). Note that the triple mutant only edits the cytidine at the fifth position, not the sixth.

0115<figref idref="DRAWINGS">FIG. 71</figref> is a graph showing the effect of various mutations on the HEK2 site with a limited number of cytidines. The spacer used was: GAAC₄AC₆AAAGCATAGACTGCGGG (SEQ ID NO: 704).

0116<figref idref="DRAWINGS">FIG. 72</figref> shows on-target base editing efficiencies of BE3 and BE3 comprising mutations W90Y R132E in immortalized astrocytes.

0117<figref idref="DRAWINGS">FIG. 73</figref> depicts a schematic of three Cpf1 fusion constructs.

0118<figref idref="DRAWINGS">FIG. 74</figref> shows a comparison of plasmid delivery of BE3 and HF-BE3 (EMX1, FANCF, and RNF2).

0119<figref idref="DRAWINGS">FIG. 75</figref> shows a comparison of plasmid delivery of BE3 and HF-BE3 (HEK3 and HEK 4).

0120<figref idref="DRAWINGS">FIGS. 76A to 76B</figref> shows off-target editing of EMX-1 at all 10 sites. This figure depicts SEQ ID NOs: 127 and 637-645

0121<figref idref="DRAWINGS">FIG. 77</figref> shows deaminase protein lipofection to HEK cells using a GAGTCCGAGCAGAAGAAGAAG (SEQ ID NO: 705) spacer. The EMX-1 on-target and EMX-1 off target site 2 were examined.

0122<figref idref="DRAWINGS">FIG. 78</figref> shows deaminase protein lipofection to HEK cells using a GGAATCCCTTCTGCAGCACCTGG (SEQ ID NO: 706) spacer. The FANCF on target and FANCF off target site 1 were examined.

0123<figref idref="DRAWINGS">FIG. 79</figref> shows deaminase protein lipofection to HEK cells using a GGCCCAGACTGAGCACGTGA (SEQ ID NO: 707) spacer. The HEK-3 on target site was examined.

0124<figref idref="DRAWINGS">FIGS. 80A to 80B</figref> show deaminase protein lipofection to HEK cells using a GGCACTGCGGCTGGAGGTGGGGG (SEQ ID NO: 708) spacer. The HEK-4 on target, off target site 1, site 3, and site 4.

0125<figref idref="DRAWINGS">FIG. 81</figref> shows the results of an in vitro assay for sgRNA activity for sgHR_13 (GTCAGGTCGAGGGTTCTGTC (SEQ ID NO: 709) spacer; C8 target: G51 to STOP), sgHR_14 (GGGCCGCAGTATCCTCACTC (SEQ ID NO: 710) spacer; C7 target; C7 target: Q68 to STOP), and sgHR_15 (CCGCCAGTCCCAGTACGGGA (SEQ ID NO: 711) spacer; C10 and C11 are targets: W239 or W237 to STOP).

0126<figref idref="DRAWINGS">FIG. 82</figref> shows the results of an in vitro assay for sgHR_17 (CAACCACTGCTCAAAGATGC (SEQ ID NO: 712) spacer; C4 and C5 are targets: W410 to STOP), and sgHR_16 (CTTCCAGGATGAGAACACAG (SEQ ID NO: 713) spacer; C4 and C5 are targets: W273 to STOP).

0127<figref idref="DRAWINGS">FIG. 83</figref> shows the direct injection of BE3 protein complexed with sgHR_13 in zebrafish embryos.

0128<figref idref="DRAWINGS">FIG. 84</figref> shows the direct injection of BE3 protein complexed with sgHR_16 in zebrafish embryos.

0129<figref idref="DRAWINGS">FIG. 85</figref> shows the direct injection of BE3 protein complexed with sgHR_17 in zebrafish embryos.

0130<figref idref="DRAWINGS">FIG. 86</figref> shows exemplary nucleic acid changes that may be made using base editors that are capable of making a cytosine to thymine change.

0131<figref idref="DRAWINGS">FIG. 87</figref> shows an illustration of apolipoprotein E (APOE) isoforms, demonstrating how a base editor (e.g., BE3) may be used to edit one APOE isoform (e.g., APOE4) into another APOE isoform (e.g., APOE3r) that is associated with a decreased risk of Alzheimer's disease.

0132<figref idref="DRAWINGS">FIG. 88</figref> shows base editing of APOE4 to APOE3r in mouse astrocytes. This figure depicts SEQ ID Nos: 671 and 627.

0133<figref idref="DRAWINGS">FIG. 89</figref> shows base editing of PRNP to cause early truncation of the protein at arginine residue 37. This figure depicts SEQ ID Nos: 577 and 714.

0134<figref idref="DRAWINGS">FIGS. 90A to 90D</figref> shows that knocking out UDG (which UGI inhibits) dramatically improves the cleanliness of efficiency of C to T base editing.

0135<figref idref="DRAWINGS">FIGS. 91A to 91B</figref> shows that use of a base editor with the nickase but without UGI leads to a mixture of outcomes, with very high indel rates.

0136<figref idref="DRAWINGS">FIGS. 92A to 92G</figref> show that SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, and VRER-BE3 mediate efficient base editing at target sites containing non-NGG PAMs in human cells. <figref idref="DRAWINGS">FIG. 92A</figref> shows base editor architectures using <i>S. pyogenes </i>and <i>S. aureus </i>Cas9. <figref idref="DRAWINGS">FIG. 92B</figref> shows recently characterized Cas9 variants with alternate or relaxed PAM requirements. <figref idref="DRAWINGS">FIGS. 92C and 92D</figref> show HEK293T cells treated with the base editor variants shown as described in Example 12. The percentage of total DNA sequencing reads (with no enrichment for transfected cells) with C converted to T at the target positions indicated are shown. The PAM sequence of each target tested is shown below the X-axis. The charts show the results for SaBE3 and SaKKH-BE3 at genomic loci with NNGRRT PAMs (<figref idref="DRAWINGS">FIG. 92C</figref>), SaBE3 and SaKKH-BE3 at genomic loci with NNNRRT PAMs (<figref idref="DRAWINGS">FIG. 92D</figref>), VQR-BE3 and EQR-BE3 at genomic loci with NGAG PAMs (<figref idref="DRAWINGS">FIG. 92E</figref>), and with NGAH PAMs (<figref idref="DRAWINGS">FIG. 92F</figref>), and VRER-BE3 at genomic loci with NGCG PAMs (<figref idref="DRAWINGS">FIG. 92G</figref>). Values and error bars reflect the mean and standard deviation of at least two biological replicates.

0137<figref idref="DRAWINGS">FIGS. 93A to 93C</figref> demonstrate that base editors with mutations in the cytidine deaminase domain exhibit narrowed editing windows. <figref idref="DRAWINGS">FIGS. 93A to 93C</figref> show HEK293T cells transfected with plasmids expressing mutant base editors and an appropriate sgRNA. Three days after transfection, genomic DNA was extracted and analyzed by high-throughput DNA sequencing at the indicated loci. The percentage of total DNA sequencing reads (without enrichment for transfected cells) with C changed to T at the target positions indicated are shown for the EMX1 site (SEQ ID NO: 721), HEK293 site 3 (SEQ ID NO: 719), FANCF site (SEQ ID NO: 722), HEK293 site 2 (SEQ ID NO: 720), site A (SEQ ID NO: 715), and site B (SEQ ID NO: 718) loci. <figref idref="DRAWINGS">FIG. 93A</figref> illustrates certain cytidine deaminase mutations which narrow the base editing window. See <figref idref="DRAWINGS">FIG. 98</figref> for the characterization of additional mutations. <figref idref="DRAWINGS">FIG. 93B</figref> shows the effect of cytidine deaminase mutations which effect the editing window width on genomic loci. Combining beneficial mutations has an additive effect on narrowing the editing window. <figref idref="DRAWINGS">FIG. 93C</figref> shows that YE1-BE3, YE2-BE3, EE-BE3, and YEE-BE3 effect the product distribution of base editing, producing predominantly singly-modified products in contrast with BE3. Values and error bars reflect the mean and standard deviation of at least two biological replicates.

0138<figref idref="DRAWINGS">FIGS. 94A and 94B</figref> show genetic variants from ClinVar that in principle can be corrected by the base editors developed in this work. The NCBI ClinVar database of human genetic variations and their corresponding phenotypes was searched for genetic diseases that in theory can be corrected by base editing. <figref idref="DRAWINGS">FIG. 94A</figref> demonstrates improvement in base editing targeting scope among all pathogenic T→C mutations in the ClinVar database through the use of base editors with altered PAM specificities. The white fractions denote the proportion of pathogenic T→C mutations accessible on the basis of the PAM requirements of either BE3, or BE3 together with the five modified-PAM base editors developed in this work. <figref idref="DRAWINGS">FIG. 94B</figref> shows improvement in base editing targeting scope among all pathogenic T→C mutations in the ClinVar database through the use of base editors with narrowed activity windows. BE3 was assumed to edit Cs in positions 4-8 with comparable efficiency as shown in <figref idref="DRAWINGS">FIGS. 93A to 93C</figref>. YEE-BE3 was assumed to edit with C5&gt;C6&gt;C7&gt;others preference within its activity window. The white fractions denote the proportion of pathogenic T→C mutations that can be edited BE3 without comparable editing of other Cs (left), or that can be edited BE3 or YEE-BE3 without comparable editing of other Cs (right).

0139<figref idref="DRAWINGS">FIGS. 95A to 95B</figref> show the effect of truncated guide RNAs on base editing window width. HEK293T cells were transfected with plasmids expressing BE3 and sgRNAs of different 5′ truncation lengths. The treated cells were analyzed as described in the Examples. <figref idref="DRAWINGS">FIG. 95A</figref> shows protospacer and PAM sequence (top, SEQ ID NO: 715) and cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, at a site within the EMX1 genomic locus. At this site, the base editing window was altered through the use of a 17-nt truncated gRNA. <figref idref="DRAWINGS">FIG. 95B</figref> shows protospacer and PAM sequences (top, SEQ ID NOs: 716 and 717) and cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, at sites within the HEK site 3 and site 4 genomic loci. At these sites, no change in the base editing window was observed, but a linear decrease in editing efficiency for all substrate bases as the sgRNA is truncated was noted.

0140<figref idref="DRAWINGS">FIG. 96</figref> shows the effect of APOBEC1-Cas9 linker lengths on base editing window width. HEK293T cells were transfected with plasmids expressing base editors with rAPOBEC1-Cas9 linkers of XTEN, GGS, (GGS)₃(SEQ ID NO: 610), (GGS)₅(SEQ ID NO: 610), or (GGS)₇(SEQ ID NO: 610) and an sgRNA. The treated cells were analyzed as described in the Examples. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown for the various base editors with different linkers.

0141<figref idref="DRAWINGS">FIGS. 97A to 97C</figref> show the effect of rAPOBEC mutations on base editing window width. <figref idref="DRAWINGS">FIG. 97C to 97D</figref> shows HEK293T cells transfected with plasmids expressing an sgRNA targeting either Site A or Site B and the BE3 point mutants indicated. The treated cells were analyzed as described in the Examples. All C's in the protospacer and within three basepairs of the protospacer are displayed and the cellular C to T conversion percentages are shown. The ‘editing window widths’, defined as the calculated number of nucleotides within which editing efficiency exceeds the half-maximal value, are displayed for all tested mutants.

0142<figref idref="DRAWINGS">FIG. 98</figref> shows the effect of APOBEC1 mutation son product distributions of base editing in mammalian cells. HEK293T cells were transfected with plasmids expressing BE3 or its mutants and an appropriate sgRNAs. The treated cells were analyzed as described in the Examples. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown (left). Percent of total sequencing reads containing the C to T conversion is shown on the right. The BE3 point mutants do not significantly affect base editing efficiencies at HEK site 4, a site with only one target cytidine.

0143<figref idref="DRAWINGS">FIG. 99</figref> shows a comparison of on-target editing plasma delivery in BE3 and HF-BE3.

0144<figref idref="DRAWINGS">FIG. 100</figref> shows a comparison of on-target editing in protein and plasma delivery of BE3.

0145<figref idref="DRAWINGS">FIG. 101</figref> shows a comparison of on-target editing in protein and plasma delivery of HF-BE3.

0146<figref idref="DRAWINGS">FIG. 102</figref> shows that both lipofection and installing HF mutations decrease off-target deamination events. The diamond indicates no off targets were detected and the specificity ratio was set to 100.

0147<figref idref="DRAWINGS">FIG. 103</figref> shows in vitro C to T editing on a synthetic substrate with Cs placed at even positions in the protospacer (NNNNTC₂TC₄TC₆TC₈TC₁₀TC₁₂TC₁₄TC₁₆TC₁₈TC₂₀NGG, SEQ ID NO: 723).

0148<figref idref="DRAWINGS">FIG. 104</figref> shows in vitro C to T editing on a synthetic substrate with Cs placed at odd positions in the protospacer (NNNNTC₂TC₄TC₆TC₈TC₁₀TC₁₂TC₁₄TC₁₆TC₁₈TC₂₀NGG, SEQ ID NO: 723).

0149<figref idref="DRAWINGS">FIG. 105</figref> includes two graphs depicting the specificity ratio of base editing with plasmid vs. protein delivery.

0150<figref idref="DRAWINGS">FIGS. 106A to 106B</figref> shows BE3 activity on non-NGG PAM sites. HEK293T cells were transfected with plasmids expressing BE3 and appropriate sgRNA. The treated cells were analyzed as described in the Examples. <figref idref="DRAWINGS">FIG. 106A</figref> shows BE3 activity on sites can be efficiently targeted by SaBE3 or SaKKH-BE3. BE3 shows low but significant activity on the NAG PAM. This figure depicts SEQ ID NOs: 728 and 729. <figref idref="DRAWINGS">FIG. 106B</figref> shows BE3 has significantly reduced editing at sites with NGA or NGCG PAMs, in contrast to VQR-BE3 or VRER-BE3. This figure depicts SEQ ID NOs: 730 and 731.

0151<figref idref="DRAWINGS">FIGS. 107A to 107B</figref> show the effect of APOBEC1 mutations on VQR-BE3 and SaKKH-BE3. HEK293T cells were transfected with plasmids expressing VQR-BE3, SaKKH-BE3 or its mutants and an appropriate sgRNAs. The treated cells were analyzed as described in the Examples below. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown. <figref idref="DRAWINGS">FIG. 107A</figref> shows that the window-modulating mutations can be applied to VQR-BE3 to enable selective base editing at sites targetable by NGA PAM. This figure depicts SEQ ID NOs: 732 and 733. <figref idref="DRAWINGS">FIG. 107B</figref> shows that, when applied to SaKKH-BE3, the mutations cause overall decrease in base editing efficiency without conferring base selectivity within the target window. This figure depicts SEQ ID NOs: 728 and 734.

0152<figref idref="DRAWINGS">FIG. 108</figref> shows a schematic representation of nucleotide editing. The following abbreviations are used: (MMR)—mismatch repair, (BE3 Nickase)—refers to base editor 3, which comprises a Cas9 nickase domain, (UGI)—uracil glycosylase inhibitor, (UDG)—uracil DNA glycosylase, (APOBEC)—refers to an APOBEC cytidine deaminase.

0153<figref idref="DRAWINGS">FIG. 109</figref> shows schematic representations of exemplary base editing constructs. The structural arrangement of base editing constructs is shown for BE3, BE4-pmCDA1, BE4-hAID, BE4-3G, BE4-N, BE4-SSB, BE4-(GGS)₃, BE4-XTEN, BE4-32aa, BE4-2×UGI, and BE4. Linkers are shown in grey (XTEN, SGGS (SEQ ID NO: 606), (GGS)₃(SEQ ID NO: 610), and 32aa). Deaminases are shown (rAPOBEC1, pmCDA1, hAID, and hAPOBEC3G). Uracil DNA Glycosylase Inhibitor (UGI) is shown. Single-stranded DNA binding protein (SSB) is shown in purple. Cas9 nickase, dCas9(A840H), is shown in red. <figref idref="DRAWINGS">FIG. 109</figref> also shows the following target sequences: EMX1, FANCF, HEK2, HEK3, HEK4, and RNF2. The amino acid sequences are indicated in SEQ ID NOs: 127-132 from top to bottom. The PAM sequences are the last three nucleotides. The target cytosine (C) is numbered and indicated in red.

0154<figref idref="DRAWINGS">FIG. 110</figref> shows the base editing results for the indicated base editing constructs (BE3, pmCDA1 hAID, hAPOBEC3G, BE4-N, BE4-SSB, BE4-(GGS)₃, BE-XTEN, BE4-32aa, and BE4-2×UGI) on the targeted cytokine (C₅) of the EMX1 sequence, GAGTC₅CGAGCAGAAGAAGAAGGG (SEQ ID NO: 127). The total percentage of targeted cytosines (C₅) that were mutated is indicated for each base editing construct, under “C₅”. The total percentage of indels is indicated for each base editing construct, under “indel”. The proportion of mutated cytosines that were mutated to an adenine (A), guanine (G), or thymine (T) are indicated for each base editing construct in the pie chart.

0155<figref idref="DRAWINGS">FIG. 111</figref> shows the base editing results for the indicated base editing constructs (BE3, pmCDA1 hAID, hAPOBEC3G, BE4-N, BE4-SSB, BE4-(GGS)₃, BE-XTEN, BE4-32aa, and BE4-2×UGI) on the targeted cytokine (C₈) of the FANCF sequence, GGAATCCC₈TTCTGCAGCACCTGG (SEQ ID NO: 128). The total percentage of targeted cytosines (C₈) that were mutated are indicated for each base editing construct, under “C₈”. The total percentage of indels are indicated for each base editing construct, under “indel”. The proportion of mutated cytosines that were mutated to an adenine (A), guanine (G), or thymine (T) are indicated for each base editing construct in the pie chart.

0156<figref idref="DRAWINGS">FIG. 112</figref> shows the base editing results for the indicated base editing constructs (BE3, pmCDA1 hAID, hAPOBEC3G, BE4-N, BE4-SSB, BE4-(GGS)₃, BE-XTEN, BE4-32aa, and BE4-2×UGI) on the targeted cytokine (C6) of the HEK2 sequence, GAACAC₆AAAGCATAGACTGCGGG (SEQ ID NO: 129). The total percentage of targeted cytosines (C₆) that were mutated are indicated for each base editing construct, under “C₆”. The total percentage of indels are indicated for each base editing construct, under “indel”. The proportion of mutated cytosines that were mutated to an adenine (A), guanine (G), or thymine (T) are indicated for each base editing construct in the pie chart.

0157<figref idref="DRAWINGS">FIG. 113</figref> shows the base editing results for the indicated base editing constructs (BE3, pmCDA1 hAID, hAPOBEC3G, BE4-N, BE4-SSB, BE4-(GGS)₃, BE-XTEN, BE4-32aa, and BE4-2×UGI) on the targeted cytokine (C₅) of the HEK3 sequence, GGCCC₅AGACTGAGCACGTGATGG (SEQ ID NO: 130). The total percentage of targeted cytosines (Cs) that were mutated are indicated for each base editing construct, under “C₅.”. The total percentage of indels are indicated for each base editing construct, under “indel”. The proportion of mutated cytosines that were mutated to an adenine (A), guanine (G), or thymine (T) are indicated for each base editing construct in the pie chart.

0158<figref idref="DRAWINGS">FIG. 114</figref> shows the base editing results for the indicated base editing constructs (BE3, pmCDA1 hAID, hAPOBEC3G, BE4-N, BE4-SSB, BE4-(GGS)₃, BE-XTEN, BE4-32aa, and BE4-2×UGI) on the targeted cytokine (C₅) of the HEK4 sequence, GGCAC₅TGCGGCTGGAGGTCCGGG (SEQ ID NO: 131). The total percentage of targeted cytosines (Cs) that were mutated are indicated for each base editing construct, under “C₅.”. The total percentage of indels are indicated for each base editing construct, under “indel”. The proportion of mutated cytosines that were mutated to an adenine (A), guanine (G), or thymine (T) are indicated for each base editing construct in the pie chart.

0159<figref idref="DRAWINGS">FIG. 115</figref> shows the base editing results for the indicated base editing constructs (BE3, pmCDA1 hAID, hAPOBEC3G, BE4-N, BE4-SSB, BE4-(GGS)₃, BE-XTEN, BE4-32aa, and BE4-2×UGI) on the targeted cytokine (C₆) of the RNF2 sequence, GTCATC₆TTAGTCATTACCTGAGG (SEQ ID NO: 132). The total percentage of targeted cytosines (C₆) that were mutated are indicated for each base editing construct, under “C₅.”. The total percentage of indels are indicated for each base editing construct, under “indel”. The proportion of mutated cytosines that were mutated to an adenine (A), guanine (G), or thymine (T) are indicated for each base editing construct in the pie chart.

0160<figref idref="DRAWINGS">FIG. 116</figref> shows exemplary fluorescent labeled (Cy3 labeled) DNA constructs used to test for Cpf1 mutants that nick the target strand. In the DNA construct of 1, both the non-target strand (top strand) and target strand (bottom strand) are fluorescently labeled. In the DNA construct of 2, the non-target strand (top strand) is fluorescently labeled and the target strand (bottom strand) is not fluorescently labeled. In the DNA construct of 3, the non-target strand (top strand) is not fluorescently labeled and the target strand (bottom strand) is fluorescently labeled.

0161<figref idref="DRAWINGS">FIG. 117</figref> shows data demonstrating the ability of various Cpf1 constructs (e.g., R836A, R1138A, wild-type) to cleave the target and non-target strands of the DNA constructs shown in <figref idref="DRAWINGS">FIG. 116</figref> over the reaction time of either 30 minutes (30 min) or greater than two hours (2 h+).

0162<figref idref="DRAWINGS">FIG. 118</figref> shows data demonstrating that a base editor having the architecture, APOBEC-AsCpf1(R912A)-UGI is capable of editing C residues (e.g., of target sequences FANCF1, FANCF2, HEK3-3, and HEK3-4) having a window from the 7^thto the 11^thbase of the target sequence. BG indicates background mutation levels (untreated). AsCpf1 indicates AsCpf1 only treated (control), APOBEC-AsCpf1(R912A)-UGI indicates a base editor containing a Cpf1 that preferentially cuts the target strand, and APOBEC-AsCpf1(R1225A)-UGI indicates a self-defeating base editor containing a Cpf1 that cuts the non-target strand. The target sequences of FANCF1, FANCF2, HEK3-3, and HEK3-4 are as follows:

0163<tables id="TABLE-US-00001" num="00001"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="56pt" align="left" /><colspec colname="2" colwidth="161pt" align="left" /><tbody valign="top"><row><entry /><entry>FANCF1</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 724)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="56pt" align="left" /><colspec colname="2" colwidth="161pt" align="left" /><tbody valign="top"><row><entry /><entry>GCGGATGTTCCAATCAGTACGCA</entry></row><row><entry></entry></row><row><entry /><entry>FANCF2</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 725)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="56pt" align="left" /><colspec colname="2" colwidth="161pt" align="left" /><tbody valign="top"><row><entry /><entry>CGAGCTTCTGGCGGTCTCAAGCA</entry></row><row><entry></entry></row><row><entry /><entry>HEK3-3</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 726)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="56pt" align="left" /><colspec colname="2" colwidth="161pt" align="left" /><tbody valign="top"><row><entry /><entry>TGCTTCTCCAGCCCTGGCCTGG</entry></row><row><entry></entry></row><row><entry /><entry>HEK3-4</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 727)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="56pt" align="left" /><colspec colname="2" colwidth="161pt" align="left" /><tbody valign="top"><row><entry /><entry>AGACTGAGCACGTGATGGCAGAG</entry></row></tbody></tgroup></table></tables>

0164<figref idref="DRAWINGS">FIG. 119</figref> shows a schematic representation of a base editor comprising a Cpf1 protein (e.g., AsCpf1 or LbCpf1). Different linker sequences (e.g., XTEN, GGS, (GGS)₃(SEQ ID NO: 610), (GGS)₅(SEQ ID NO: 610), and (GGS)₇(SEQ ID NO: 610)) were tested for the portion labeled “linker,” results of which are shown in <figref idref="DRAWINGS">FIG. 120</figref>.

0165<figref idref="DRAWINGS">FIG. 120</figref> shows data demonstrating the ability of the construct shown in <figref idref="DRAWINGS">FIG. 119</figref> to edit the Cs residue of the HEK3 site TGCTTCTC₈CAGCCCTGGCCTGG (SEQ ID NO: 592). Different linker sequences, which link the APOBEC domain to the Cpf1 domain (e.g., LbCpf1(R836A) or AsCpf1(R912A)) were tested. Exemplary linkers that were tested include XTEN, GGS, (GGS)₃(SEQ ID NO: 610), (GGS)₅(SEQ ID NO: 610), and (GGS)₇(SEQ ID NO: 610).

0166<figref idref="DRAWINGS">FIG. 121</figref> shows data demonstrating the ability of the construct shown in <figref idref="DRAWINGS">FIG. 119</figref>, having the LbCpf1 domain, to edit the C₈and C₉residues of the HEK3 TGCTTCTC₈C₉AGCCCTGGCCTGG (SEQ ID NO: 592). Different linker sequences from a database maintained by the Centre of Integrative Bioinformatics VU, which link the APOBEC domain to the LbCpf1 domain were tested. Exemplary linkers that were tested include 1au7, 1c1k, 1c20, 1ee8, 1flz, 1ign, 1jmc, 1sfe, 2ezx, and 2reb.

0167<figref idref="DRAWINGS">FIG. 122</figref> shows a schematic representation of the structure of AsCpf1, where the N and C termini are indicated.

0168<figref idref="DRAWINGS">FIG. 123</figref> shows a schematic representation of the structure of SpCas9, where the N and C termini are indicated.

0169<figref idref="DRAWINGS">FIG. 124</figref> shows a schematic representation of AsCpf1, where the red circle indicates the predicted area where the editing window is. The square indicates a helical region that may be obstructing APOBEC activity.

0170<figref idref="DRAWINGS">FIGS. 125A and 125B</figref> show engineering and in vitro characterization of a high fidelity base editor (HF-BE3). <figref idref="DRAWINGS">FIG. 125A</figref> shows a schematic representation of HF-BE3. Point mutations introduced into BE3 to generate HF-BE3 are shown. The representation used PDB structures 4UN3 (Cas9), 4ROV (cytidine deaminase) and 1UGI (uracil DNA glycosylase inhibitor). <figref idref="DRAWINGS">FIG. 125B</figref> shows in vitro deamination of synthetic substrates containing “TC” repeat protospacers. Values and error bars reflect mean and range of two independent replicates performed on different days.

0171<figref idref="DRAWINGS">FIGS. 126A to 126C</figref> show purification of base editor proteins. <figref idref="DRAWINGS">FIG. 126A</figref> shows selection of optimal <i>E. coli </i>strain for base editor expression. After IPTG-induced protein expression for 16 h at 18° C., crude cell lysate was analyzed for protein content. BL21 Star (DE3) (Thermo Fisher) cells showed the most promising post-expression levels of both BE3 and HF-BE3 and were used for expression of base editors. <figref idref="DRAWINGS">FIG. 126B</figref> shows purification of expressed base editor proteins. Placing the His6 tag on the C-terminus of the base editors lead to production of a truncation product for both BE3 and HF-BE3 (lanes 1 and 2). Unexpectedly, this truncation product was removed by placing the His6 tag on the N-terminus of the protein (lanes 3-6). Inducing expression of base editors at a cell density of OD600=0.7 (lanes 4-5), later than is optimal for Cas9 expression (OD600=0.4)1, improves yield of base editor proteins. Purification was performed using a manual HisPur resin column followed by cation exchange FPLC (Akta). <figref idref="DRAWINGS">FIG. 126C</figref> shows purified BE3 and HF-BE3. Different concentrations of purified BE3 and HF-BE3 were denatured using heat and LDS and loaded onto a polyacrylamide gel. Protein samples are representative of proteins used in this study. Gels in <figref idref="DRAWINGS">FIGS. 126A to 126C</figref> are BOLT Bis-Tris Plus 4-12% polyacrylamide (Thermo Fisher). Electrophoresis and staining were performed as described in Methods.

0172<figref idref="DRAWINGS">FIGS. 127A to 127D</figref> show activity of a high fidelity base editor (HF-BE3) in human cells. <figref idref="DRAWINGS">FIGS. 127A to 127C</figref> show on- and off-target editing associated with plasmid transfection of BE3 and HF-BE3 was assayed using high-throughput sequencing of genomic DNA from HEK293T cells treated with sgRNAs targeting non-repetitive genomic loci EMX1 (<figref idref="DRAWINGS">FIG. 127A</figref>), FANCF (<figref idref="DRAWINGS">FIG. 127B</figref>), and HEK293 site 3 (<figref idref="DRAWINGS">FIG. 127C</figref>). On- and off-target loci associated with each sgRNA are separated by a vertical line. <figref idref="DRAWINGS">FIG. 127D</figref> shows on- and off-target editing associated with the highly repetitive sgRNA targeting VEGFA site 2. Values and error bars reflect mean±S.D. of three independent biological replicates performed on different days. For <figref idref="DRAWINGS">FIGS. 127A to 127C</figref>, stars indicate significant editing based on a comparison between the treated sample and an untreated control. * p≤0.05, ** p≤0.01 and *** p≤0.001 (Student's two tailed t-test). For <figref idref="DRAWINGS">FIG. 127D</figref>, asterisks are not shown since all treated samples displayed significant editing relative to the control. Individual p-values are listed in in Table 16.

0173<figref idref="DRAWINGS">FIGS. 128A to 128C</figref> show the effect of dosage of BE3 protein or plasmid on the efficiency of on-target and off-target base editing in cultured human cells. <figref idref="DRAWINGS">FIG. 128A</figref> shows on-target editing efficiency at each of the four genomic loci was averaged across all edited cytosines in the activity window for each sgRNA. Values and error bars reflect mean±S.E.M of three independent biological replicates performed on different days. <figref idref="DRAWINGS">FIGS. 128B and 128C</figref> show on- and off-target editing at the EMX1 site arising from BE3 plasmid titration (<figref idref="DRAWINGS">FIG. 128B</figref>) or BE3 protein titration (<figref idref="DRAWINGS">FIG. 128C</figref>) in HEK293T cells. Values and error bars reflect mean±S.D. of three independent biological replicates performed on different days.

0174<figref idref="DRAWINGS">FIGS. 129A to 129B</figref> show on-target:off-target base editing frequency ratios for plasmid and protein delivery of BE3 and HF-BE3. Base editing on-target:off-target specificity ratios were calculated by dividing the on-target editing percentage at a particular cytosine in the activity window by the off-target editing percentage at the corresponding cytosine for the indicated off-target locus (see Methods). When off-target editing was below the threshold of detection (0.025% of sequencing reads), we set the off-target editing to the limit of detection (0.025%) and divided the on-target editing percentage by this upper limit. In these cases, denoted by ♦, the specificity ratios shown represent lower limits. Specificity ratios are shown for non-repetitive sgRNAs FANCF, HEK 293 site 3, and FANCF (<figref idref="DRAWINGS">FIG. 129A</figref>) and for the highly repetitive sgRNA VEGFA site 2 (<figref idref="DRAWINGS">FIG. 129B</figref>). Values and error bars reflect mean±S.D. of three independent biological replicates performed on different days.

0175<figref idref="DRAWINGS">FIGS. 130A to 130D</figref> show protein delivery of base editors into cultured human cells. <figref idref="DRAWINGS">FIGS. 130A to 130D</figref> show on- and off-target editing associated with RNP delivery of base editors complexed with sgRNAs targeting EMX1 (<figref idref="DRAWINGS">FIG. 130A</figref>), FANCF (<figref idref="DRAWINGS">FIG. 130B</figref>), HEK293 site 3 (<figref idref="DRAWINGS">FIG. 130C</figref>) and VEGFA site 2 (<figref idref="DRAWINGS">FIG. 130D</figref>). Off-target base editing was undetectable at all of the sequenced loci for non-repetitive sgRNAs. Values and error bars reflect mean±S.D. of three independent biological replicates performed on different days. Stars indicate significant editing based on a comparison between the treated sample and an untreated control. * p≤0.05, ** p≤0.01 and *** p≤0.001 (Student's two tailed t-test).

0176<figref idref="DRAWINGS">FIGS. 131A to 131C</figref> show indel formation associated with base editing at genomic loci. <figref idref="DRAWINGS">FIG. 131A</figref> shows indel frequency at on-target loci for VEGFA site 2, EMX1, FANCF, and HEK293 site 3 sgRNAs. <figref idref="DRAWINGS">FIG. 131B</figref> shows the ratio of base editing:indel formation. The diamond (♦) indicates no indels were detected (no significant difference in indel frequency in the treated sample and in the untreated control). <figref idref="DRAWINGS">FIG. 131C</figref> shows indels observed at the off-target loci associated with the on-target sites interrogated in <figref idref="DRAWINGS">FIG. 131A</figref>. Values and error bars reflect mean±S.D. of three independent biological replicates performed on different days.

0177<figref idref="DRAWINGS">FIGS. 132A to 132D</figref> show DNA-free in vivo base editing in zebrafish embryos and in the inner ear of live mice using RNP delivery of BE3. <figref idref="DRAWINGS">FIG. 132A</figref> shows on-target genome editing in zebrafish harvested 4 days after injection of BE3 complexed with indicated sgRNA. Values and error bars reflect mean±s.d. of three injected and three control zebrafish. Controls were injected with BE3 complexed with an unrelated sgRNA. <figref idref="DRAWINGS">FIG. 132B</figref> shows schematic showing in vivo injection of BE3:sgRNA complexes encapsulated into cationic lipid nanoparticles <figref idref="DRAWINGS">FIG. 132C</figref> shows base editing of cytosine residues in the base editor window at the VEGFA site 2 genomic locus. <figref idref="DRAWINGS">FIG. 132D</figref> shows on-target editing at each cytosine in the base editing window of the VEGFA site 2 target locus. <figref idref="DRAWINGS">FIG. 132D</figref> (<figref idref="DRAWINGS">FIGS. 132C and 132D</figref>) shows values and error bars reflect mean±S.E.M. of three mice injected with sgRNA targeting VEGFA Site 2, three uninjected mice and one mouse injected with unrelated sgRNA.

0178<figref idref="DRAWINGS">FIGS. 133A to 133E</figref> show on- and off-target base editing in murine NIH/3T3 cells. <figref idref="DRAWINGS">FIG. 133A</figref> shows on-target base editing associated with the ‘VEGFA site 2’ sgRNA (See <figref idref="DRAWINGS">FIG. 132E</figref> for sequences). The negative control corresponds to cells treated with plasmid encoding BE3 but no sgRNA. Values and error bars reflect mean±S.D. of three independent biological replicates performed on different days. <figref idref="DRAWINGS">FIGS. 133B to 133E</figref> show off-target editing associated with this site was measured using high-throughput DNA sequencing at the top four predicted off-target loci for this sgRNA (sequences shown in <figref idref="DRAWINGS">FIG. 132E</figref>). <figref idref="DRAWINGS">FIG. 133B</figref> shows off-target 2, <figref idref="DRAWINGS">FIG. 133C</figref> shows off-target 1, <figref idref="DRAWINGS">FIG. 133D</figref> shows off-target 3, <figref idref="DRAWINGS">FIG. 133E</figref> shows off-target 4. Values and error bars reflect mean±S.D. of three independent biological replicates performed on different days.

0179<figref idref="DRAWINGS">FIGS. 134A to 134B</figref> show off-target base editing and on-target indel analysis from in vivo-edited murine tissue. <figref idref="DRAWINGS">FIG. 134A</figref> shows editing plotted for each cytosine in the base editing window of off-target loci associated with VEGFA site 2. <figref idref="DRAWINGS">FIG. 134B</figref> shows indel rates at the on-target base editor locus. Values and error bars reflect mean±S.E.M of three injected and three control mice.

0180<figref idref="DRAWINGS">FIGS. 135A to 135C</figref> show the effects on base editing product purity of knocking out UNG. <figref idref="DRAWINGS">FIG. 135A</figref> shows HAP1 (UNG⁺) and HAP1 UNG⁻ cells treated with BE3 as described in the Materials and Methods of Example 17. The product distribution among edited DNA sequencing reads (reads in which the target C is mutated) is shown. <figref idref="DRAWINGS">FIG. 135B</figref> shows protospacers and PAM sequences of the genomic loci tested, with the target Cs analyzed in <figref idref="DRAWINGS">FIG. 135A</figref> shown in red. <figref idref="DRAWINGS">FIG. 135C</figref> shows the frequency of indel formation following treatment with BE3 in HAP1 cells or HAP1 UNG⁻ cells. Values and error bars reflect the mean±S.D. of three independent biological replicates performed on different days.

0181<figref idref="DRAWINGS">FIGS. 136A to 136D</figref> show the effects of multi-C base editing on product purity. <figref idref="DRAWINGS">FIG. 136A</figref> shows representative high-throughput sequencing data of untreated, BE3-treated, and AID-BE3-treated human HEK293T cells. The sequence of the protospacer is shown at the top, with the PAM and the target Cs in red with subscripted numbers indicating their position within the protospacer. Underneath each sequence are the percentages of total sequencing reads with the corresponding base. The relative percentage of target Cs that are cleanly edited to T rather than to non-T bases is much higher for AID-BE3-treated cells, which edits three Cs at this locus, than for BE3-treated cells, which edits only one C. <figref idref="DRAWINGS">FIG. 136B</figref> shows HEK293T cells treated with BE3, CDA1-BE3, and AID-BE3 as described in the Materials and Methods of Example 17. The product distribution among edited DNA sequencing reads (reads in which the target C is mutated) is shown. <figref idref="DRAWINGS">FIG. 136C</figref> shows protospacers and PAM sequences of genomic loci studied, with the target Cs that are analyzed in <figref idref="DRAWINGS">FIG. 136B</figref> shown in red. <figref idref="DRAWINGS">FIG. 136D</figref> shows the frequency of indel formation following the treatment shown in <figref idref="DRAWINGS">FIG. 136A</figref>. Values and error bars reflect the mean±S.D. of three independent biological replicates performed on different days.

0182<figref idref="DRAWINGS">FIGS. 137A to 137C</figref> show the effects on C-to-T editing efficiencies and product purities of changing the architecture of BE3. <figref idref="DRAWINGS">FIG. 137A</figref> shows protospacers and PAM sequences of genomic loci studied, with the target Cs in <figref idref="DRAWINGS">FIG. 137C</figref> shown in purple and red, and the target Cs in <figref idref="DRAWINGS">FIG. 137B</figref> shown in red. <figref idref="DRAWINGS">FIG. 137B</figref> shows HEK293T cells treated with BE3, SSB-BE3, N-UGI-BE3, and BE3-2×UGI as described in the Materials and Methods of Example 17. The product distribution among edited DNA sequencing reads (reads in which the target C is mutated) is shown for BE3, N-UGI-BE3, and BE3-2×UGI. <figref idref="DRAWINGS">FIG. 137C</figref> shows C-to-T base editing efficiencies. Values and error bars reflect the mean±S.D. of three independent biological replicates performed on different days.

0183<figref idref="DRAWINGS">FIGS. 138A to 138D</figref> show the effects of linker length variation in BE3 on C-to-T editing efficiencies and product purities. <figref idref="DRAWINGS">FIG. 138A</figref> shows the architecture of BE3, BE3C, BE3D, and BE3E <figref idref="DRAWINGS">FIG. 138B</figref> shows protospacers and PAM sequences of genomic loci studied, with the target Cs in <figref idref="DRAWINGS">FIG. 138C</figref> shown in purple and red, and target Cs in <figref idref="DRAWINGS">FIG. 138D</figref> shown in red. <figref idref="DRAWINGS">FIG. 138C</figref> shows HEK293T cells treated with BE3, BE3C, BE3D, or BE3E as described in the Materials and Methods of Example 17. C-to-T base editing efficiencies are shown. <figref idref="DRAWINGS">FIG. 138D</figref> shows the product distribution among edited DNA sequencing reads (reads in which the target C is mutated) for BE3, BE3C, BE3D, and BE3E. Values and error bars reflect the mean±S.D. of three independent biological replicates performed on different days.

0184<figref idref="DRAWINGS">FIGS. 139A to 139D</figref> show BE4 increases base editing efficiency and product purities compared to BE3. <figref idref="DRAWINGS">FIG. 139A</figref> shows the architectures of BE3, BE4, and Target-AID. <figref idref="DRAWINGS">FIG. 139B</figref> shows protospacers and PAM sequences of genomic loci studied, with the target Cs in <figref idref="DRAWINGS">FIG. 139C</figref> shown in purple and red, and the target Cs in <figref idref="DRAWINGS">FIG. 139D</figref> shown in red. <figref idref="DRAWINGS">FIG. 139C</figref> shows HEK293T cells treated with BE3, BE4, or Target-AID as described in the Materials and Methods of Example 17. C-to-T base editing efficiencies are shown. <figref idref="DRAWINGS">FIG. 139D</figref> shows the product distribution among edited DNA sequencing reads (reads in which the target C is mutated) for BE3 and BE4. Values and error bars reflect the mean±S.D. of three independent biological replicates performed on different days.

0185<figref idref="DRAWINGS">FIGS. 140A to 140C</figref> show CDA1-BE3 and AID-BE3 edit Cs following target Gs more efficiently than BE3. <figref idref="DRAWINGS">FIG. 140A</figref> shows protospacer and PAM sequences of genomic loci studied, with target Cs edited by BE3, CDA1-BE3, and AID-BE3 shown in red, and target Cs (following Gs) edited by CDA1-BE3 and AID-BE3 only shown in purple. <figref idref="DRAWINGS">FIG. 140B</figref> shows HEK293T cells treated with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 as described in the Materials and Methods of Example 17. C-to-T base editing efficiencies are shown. <figref idref="DRAWINGS">FIG. 140C</figref> shows individual DNA sequencing reads from HEK293T cells that were treated with BE3, CDA1-BE3, or AID-BE3 targeting the HEK2 locus and binned according to the sequence of the protospacer and analyzed, revealing that &gt;85% of sequencing reads that have clean C to T edits by CDA1-BE3 and AID-BE3 have both Cs edited to T (<figref idref="DRAWINGS">FIG. 140C</figref>).

0186<figref idref="DRAWINGS">FIGS. 141A to 141C</figref> show uneven editing in sites with multiple editable Cs results in lower product purity. <figref idref="DRAWINGS">FIG. 141A</figref> shows protospacers and PAM sequences of genomic loci studied, with the target Cs in <figref idref="DRAWINGS">FIG. 141C</figref> shown in purple and red, and target Cs in <figref idref="DRAWINGS">FIG. 141B</figref> shown in red. <figref idref="DRAWINGS">FIGS. 141B and 141C</figref> show HEK293T cells treated with BE3 as described in the Materials and Methods of Example 17. The product distribution among edited DNA sequencing reads (reads in which the target C is mutated) is shown. C to non-T editing is more frequent when editing efficiencies are unequal for two Cs within the same locus. Values and error bars reflect the mean±S.D. of three independent biological replicates performed on different days.

0187<figref idref="DRAWINGS">FIGS. 142A to 142D</figref> show base editing of multiple Cs results in higher base editing product purity. <figref idref="DRAWINGS">FIG. 142A</figref> shows protospacers and PAM sequences of genomic loci studied, with the target Cs that are investigated in <figref idref="DRAWINGS">FIG. 142B</figref> shown in red. <figref idref="DRAWINGS">FIG. 142B</figref> shows HEK293T cells treated with BE3 or BE3B (which lacks UGI) as described in the Materials and Methods of Example 17. The product distribution among edited DNA sequencing reads (reads in which the target C is mutated) is shown. <figref idref="DRAWINGS">FIG. 142C</figref> shows the HTS reads from HEK293T cells that were treated with BE3 or BE3B (which lacks UGI) targeting the HEK2 locus were binned according to the identity of the primary target C at position 6. The resulting reads were then analyzed for the identity of the base at the secondary target C at position 4. C₆is more likely to be incorrectly edited to a non-T when there is only a single editing event in that read. <figref idref="DRAWINGS">FIG. 142D</figref> shows the distribution of edited reads with A, G, and T at C₅in cells treated with BE3 or BE3B targeting the HEK4 locus (a site with only a single editable C), illustrating that single G:U mismatches are processed via UNG-initiated base excision repair to give a mixture of products. Values and error bars reflect the mean±S.D. of three independent biological replicates performed on different days.

0188<figref idref="DRAWINGS">FIG. 143</figref> shows base editing of multiple Cs results in higher base editing product purity at the HEK3 and RNF2 loci. DNA sequencing reads from HEK293T cells treated with BE3 or BE3B (without UGI) targeting the HEK3 and RNF2 loci were separated according to the identity of the base at the primary target C position (in red). The four groups of sequencing reads were then interrogated for the identity of the base at the secondary target C position (in purple). For BE3, when the primary target C (in red) is incorrectly edited to G, the secondary target C is more likely to remain C. Conversely, when the primary target C (in red) is converted to T, the secondary target C is more likely to also be edited to a T in the same sequencing read. Values and error bars reflect the mean±S.D. of three independent biological replicates performed on different days.

0189<figref idref="DRAWINGS">FIGS. 144A to 144C</figref> show BE4 induces lower indel frequencies than BE3, and Target-AID exhibits similar product purities as CDA1-BE3. <figref idref="DRAWINGS">FIG. 144A</figref> shows HEK293T cells treated with BE3, BE4, or Target-AID as described in the Materials and Methods of Example 17. The frequency of indel formation (see Materials and Methods of Example 17) is shown. <figref idref="DRAWINGS">FIG. 144B</figref> shows HEK293T cells treated with CDA1-BE3 or Target-AID as described in the Materials and Methods of Example 17. The product distribution among edited DNA sequencing reads (reads in which the target C is mutated) is shown. <figref idref="DRAWINGS">FIG. 144C</figref> shows protospacers and PAM sequences of genomic loci studied, with the target Cs that are investigated in <figref idref="DRAWINGS">FIG. 144B</figref> shown in red. Values and error bars reflect the mean±S.D. of three independent biological replicates performed on different days.

0190<figref idref="DRAWINGS">FIGS. 145A to 145C</figref> show SaBE4 exhibits increased base editing yields and product purities compared to SaBE3. <figref idref="DRAWINGS">FIG. 145A</figref> shows HEK293T cells treated with SaBE3 and SaBE4 as described in the Materials and Methods of Example 17. The percentage of total DNA sequencing reads with Ts at the target positions indicated are shown. <figref idref="DRAWINGS">FIG. 145B</figref> shows protospacers and PAM sequences of genomic loci studied, with the target Cs in <figref idref="DRAWINGS">FIG. 145A</figref> shown in purple and red, with target Cs that are investigated in <figref idref="DRAWINGS">FIG. 145C</figref> shown in red. <figref idref="DRAWINGS">FIG. 145C</figref> shows the product distribution among edited DNA sequencing reads (reads in which the target C is mutated). Values and error bars reflect the mean±S.D. of three independent biological replicates performed on different days.

0191<figref idref="DRAWINGS">FIG. 146</figref> shows base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the EMX1 locus. The sequence of the protospacer is shown at the top, with the PAM and the target bases in red with a subscripted number indicating their positions within the protospacer. Underneath the sequence are the percentages of total sequencing reads with the corresponding base. Cells were treated as described in the Materials and Methods of Example 17. Values shown are from one representative experiment.

0192<figref idref="DRAWINGS">FIG. 147</figref> shows base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the FANCF locus. The sequence of the protospacer is shown at the top, with the PAM and the target bases in red with a subscripted number indicating their positions within the protospacer. Underneath the sequence are the percentages of total sequencing reads with the corresponding base. Cells were treated as described in the Materials and Methods of Example 17. Values shown are from one representative experiment.

0193<figref idref="DRAWINGS">FIG. 148</figref> shows base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the HEK2 locus. The sequence of the protospacer is shown at the top, with the PAM and the target bases in red with a subscripted number indicating their positions within the protospacer. Underneath the sequence are the percentages of total sequencing reads with the corresponding base. Cells were treated as described in the Materials and Methods of Example 17. Values shown are from one representative experiment.

0194<figref idref="DRAWINGS">FIG. 149</figref> shows base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the HEK3 locus. The sequence of the protospacer is shown at the top, with the PAM and the target bases in red with a subscripted number indicating their positions within the protospacer. Underneath the sequence are the percentages of total sequencing reads with the corresponding base. Cells were treated as described in the Materials and Methods of Example 17. Values shown are from one representative experiment.

0195<figref idref="DRAWINGS">FIG. 150</figref> shows base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the HEK4 locus. The sequence of the protospacer is shown at the top, with the PAM and the target bases in red with a subscripted number indicating their positions within the protospacer. Underneath the sequence are the percentages of total sequencing reads with the corresponding base. Cells were treated as described in the Materials and Methods of Example 17. Values shown are from one representative experiment.

0196<figref idref="DRAWINGS">FIG. 151</figref> shows base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the RNF2 locus. The sequence of the protospacer is shown at the top, with the PAM and the target bases in red with a subscripted number indicating their positions within the protospacer. Underneath the sequence are the percentages of total sequencing reads with the corresponding base. Cells were treated as described in the Materials and Methods of Example 17. Values shown are from one representative experiment.

0197<figref idref="DRAWINGS">FIG. 152</figref> shows a schematic of LBCpf1 fusion constructs. Construct 10 has a domain arrangement of [Apobec]-[LbCpf1]-[UGI]-[UGI]; construct 11 has a domain arrangement of [Apobec]-[LbCpf1]-[UGI]; construct 12 has a domain arrangement of [UGI]-[Apobec]-[LbCpf1]; construct 13 has a domain arrangement of [Apobec]-[UGI]-[LbCpf1]; construct 14 has a domain arrangement of [LbCpf1]-[UGI]-[Apobec]; construct 15 has a domain arrangement of [LbCpf1]-[Apobec]-[UGI]. For each construct three different LbCpf1 proteins were used (D/N/A, which refers to nuclease dead LbCpf1 (D); LbCpf1 nickase (N) and nuclease active LbCpf1 (A)).

0198<figref idref="DRAWINGS">FIG. 153</figref> shows the percentage of C to T editing of six C residues in the EMX target TTTGTAC₃TTTGTC₉C₁₀TC12C₁₃GGTTC₁₈TG (SEQ ID NO: 738) using a guide of 19 nucleotides in length, i.e., EMX19: TACTTTGTCCTCCGGTTCT (SEQ ID NO: 744). Editing was tested for several of the constructs shown in <figref idref="DRAWINGS">FIG. 152</figref>.

0199<figref idref="DRAWINGS">FIG. 154</figref> shows the percentage of C to T editing of six C residues in the EMX target TTTGTAC₃TTTGTC₉C₁₀TC12C₁₃GGTTC₁₈TG (SEQ ID NO: 738) using a guide of 18 nucleotides in length, i.e., EMX18: TACTTTGTCCTCCGGTTC (SEQ ID NO: 745). Editing was tested for several of the constructs shown in <figref idref="DRAWINGS">FIG. 152</figref>.

0200<figref idref="DRAWINGS">FIG. 155</figref> shows the percentage of C to T editing of six C residues in the EMX target TTTGTAC₃TTTGTC₉C₁₀TC12C₁₃GGTTC₁₈TG (SEQ ID NO: 738) using a guide of 17 nucleotides in length, i.e., EMX17: TACTTTGTCCTCCGGTT (SEQ ID NO: 746). Editing was tested for several of the constructs shown in <figref idref="DRAWINGS">FIG. 152</figref>.

0201<figref idref="DRAWINGS">FIG. 156</figref> shows the percentage of C to T editing of eight C residues in the HEK2 target TTTCC₁AGC₄C₅C₆GC₈TGGC₁₂C₁₃C₁₄TGTAAA (SEQ ID NO: 739) using a guide of 23 nucleotides in length, i.e., Hek2_23: CAGCCCGCTGGCCCTGTAAAGGA (SEQ ID NO: 747). Editing was tested for several of the constructs shown in <figref idref="DRAWINGS">FIG. 152</figref>.

0202<figref idref="DRAWINGS">FIG. 157</figref> shows the percentage of C to T editing of eight C residues in the HEK2 target TTTCC₁AGC₄C₅C₆GC₈TGGC₁₂C₁₃C₁₄TGTAAA (SEQ ID NO: 739) using a guide of 20 nucleotides in length, i.e., Hek2_20: CAGCCCGCTGGCCCTGTAAA (SEQ ID NO: 748). Editing was tested for several of the constructs shown in <figref idref="DRAWINGS">FIG. 152</figref>.

0203<figref idref="DRAWINGS">FIG. 158</figref> shows the percentage of C to T editing of eight C residues in the HEK2 target TTTCC₁AGC₄C₅C₆GC₈TGGC₁₂C₁₃C₁₄TGTAAA (SEQ ID NO: 739) using a guide of 19 nucleotides in length, i.e., Hek2_19: CAGCCCGCTGGCCCTGTAA (SEQ ID NO: 749). Editing was tested for several of the constructs shown in <figref idref="DRAWINGS">FIG. 152</figref>.

0204<figref idref="DRAWINGS">FIG. 159</figref> shows the percentage of C to T editing of eight C residues in the HEK2 target TTTCC₁AGC₄C₅C₆GC₈TGGC₁₂C₁₃C₁₄TGTAAA (SEQ ID NO: 739) using a guide of 18 nucleotides in length, i.e., Hek2_18: CAGCCCGCTGGCCCTGTA (SEQ ID NO: 750). Editing was tested for several of the constructs shown in <figref idref="DRAWINGS">FIG. 152</figref>.

0205<figref idref="DRAWINGS">FIG. 160</figref> shows the editing percentage values (after adjustment based on indel count), and the percentage of indels for the experiments depicted in <figref idref="DRAWINGS">FIG. 153</figref>.

0206<figref idref="DRAWINGS">FIG. 161</figref> shows the editing percentage values (after adjustment based on indel count), and the percentage of indels for the experiments depicted in <figref idref="DRAWINGS">FIG. 154</figref>.

0207<figref idref="DRAWINGS">FIG. 162</figref> shows the editing percentage values (after adjustment based on indel count), and the percentage of indels for the experiments depicted in <figref idref="DRAWINGS">FIG. 155</figref>.

0208<figref idref="DRAWINGS">FIG. 163</figref> shows the editing percentage values (after adjustment based on indel count), and the percentage of indels for the experiments depicted in <figref idref="DRAWINGS">FIG. 156</figref>.

0209<figref idref="DRAWINGS">FIG. 164</figref> shows the editing percentage values (after adjustment based on indel count), and the percentage of indels for the experiments depicted in <figref idref="DRAWINGS">FIG. 157</figref>.

0210<figref idref="DRAWINGS">FIG. 165</figref> shows the editing percentage values (after adjustment based on indel count), and the percentage of indels for the experiments depicted in <figref idref="DRAWINGS">FIG. 158</figref>.

0211<figref idref="DRAWINGS">FIG. 166</figref> shows the editing percentage values (after adjustment based on indel count), and the percentage of indels for the experiments depicted in <figref idref="DRAWINGS">FIG. 159</figref>.

DEFINITIONS

0212As used herein and in the claims, the singular forms “a,” “an,” and “the” include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to “an agent” includes a single agent and a plurality of such agents.

0213The term “nucleic acid programmable DNA binding protein” or “napDNAbp” refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid (e.g., gRNA), that guides the napDNAbp to a specific nucleic acid sequence, for example, by hybridinzing to the target nucleic acid sequence. For example, a Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence is has complementary to the guide RNA. In some embodiments, the napDNAbp is a class 2 microbial CRISPR-Cas effector. In some embodiments, the napDNAbp is a Cas9 domain, for example, a nuclease active Cas9, a Cas9 nickase (nCas9), or a nuclease inactive Cas9 (dCas9). Examples of nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute. It should be appreciated, however, that nucleic acid programmable DNA binding proteins also include nucleic acid programmable proteins that bind RNA. For example, the napDNAbp may be associated with a nucleic acid that guides the napDNAbp to an RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, though they may not be specifically described in this disclosure.

0214In some embodiments, the napDNAby is an “RNA-programmable nuclease” or “RNA-guided nuclease.” The terms are used interchangeably herein and refer to a nuclease that forms a complex with (e.g., binds or associates with) one or more RNA(s) that is not a target for cleavage. In some embodiments, an RNA-programmable nuclease, when in a complex with an RNA, may be referred to as a nuclease:RNA complex. Typically, the bound RNA(s) is referred to as a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though “gRNA” is also used to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules. Typically, gRNAs that exist as a single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (i.e., directs binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as a tracrRNA and comprises a stem-loop structure. In some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., <i>Science </i>337:816-821 (2012), the entire contents of which is incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in U.S. Provisional Patent Application, U.S. Ser. No. 61/874,682, filed Sep. 6, 2013, entitled “Switchable Cas9 Nucleases And Uses Thereof,” and U.S. Provisional Patent Application, U.S. Ser. No. 61/874,746, filed Sep. 6, 2013, entitled “Delivery System For Functional Nucleases,” the entire contents of each are hereby incorporated by reference in their entirety. In some embodiments, a gRNA comprises two or more of domains (1) and (2), and may be referred to as an “extended gRNA.” For example, an extended gRNA will bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions, as described herein. The gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site, providing the sequence specificity of the nuclease:RNA complex. In some embodiments, the RNA-programmable nuclease is the (CRISPR-associated system) Cas9 endonuclease, for example, Cas9 (Csn1) from <i>Streptococcus pyogenes </i>(see, e.g., “Complete genome sequence of an M1 strain of <i>Streptococcus pyogenes</i>.” Ferretti J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607 (2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference.

0215In some embodiments, any of the sgRNAs provided herein comprise a sequence, e.g., a sgRNA backbone sequence that binds to a napDNAbp. For example sgRNAs have been described in Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J A, and Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337, 816-812; Mali P, Esvelt K M, Church G M (2013) Cas9 as a versatile tool for engineering biology. Nature Methods, 10, 957-963; Li J F, Norville J E, Aach J, McCromack M, Zhang D, Bush J, Church G M, and Sheen J (2013) Multiplex and homologous recombination-mediated genome editing in <i>Arabidopsis </i>and <i>Nicotiana benthamiana </i>using guide RNA and Cas9. Nature Biotech, 31, 688-691; Hwang W Y, Fu Y, Reyon D, Maeder M L, Tsai S Q, Sander J D, Peterson R T, Yeh J R J, Joung J K (2013) Efficient in vivo genome editing using RNA-guided nucleases. Nat Biotechnol, 31, 227-229; Cong L, Ran F A, Cox D, Lin S, Barretto R, Habib N, Hsu P D, Wu X, Jiang W, Marraffini L A, Zhang F (2013) Multiplex genome engineering using CRIPSR/Cas systems. Science, 339, 819-823; Cho S W, Kim S, Kim J M, Kim J S (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol, 31, 230-232; Jinek M J, East A, Cheng A, Lin S, Ma E, Doudna J (2013) RNA-programmed genome editing in human cells. eLIFE, 2:e00471; DiCarlo J E, Norville J E, Mali P, Rios, Aach J, Church G M (2013) Genome engineering in <i>Saccharomyces cerevisiae </i>using CRISPR-Cas systems. Nucl Acids Res, 41, 4336-4343; Briner A E, Donohoue P D, Gomaa A A, Selle K, Slorach E M, Nye C H, Haurwitz R E, Beisel C L, May A P, and Barrangou R (2014) Guide RNA functional modules direct Cas9 activity and orthogonality. Mol Cell, 56, 333-339; the contents of each of which are incorporated herein by reference. In some embodiments, any of the gRNAs (e.g., sgRNAs) provided herein comprise the nucleic acid sequence of GTAATTTCTACTAAGTGTAGAT (SEQ ID NO: 741), wherein each of the Ts of SEQ ID NO: 741 are uracil (U), i.e., GUAAUUUCUACUAAGUGUAGAU, or the sequence GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG AAAAAGUGGCACCGAGUCGGUGCUUUUU-3′ (SEQ ID NO: 618).

0216Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins are able to target, in principle, any sequence specified by the guide RNA. Methods of using RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong, L. et al., Multiplex genome engineering using CRISPR/Cas systems. <i>Science </i>339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9<i>. Science </i>339, 823-826 (2013); Hwang, W. Y. et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. <i>Nature biotechnology </i>31, 227-229 (2013); Jinek, M. et al. RNA-programmed genome editing in human cells. <i>eLife </i>2, e00471 (2013); Dicarlo, J. E. et al., Genome engineering in <i>Saccharomyces cerevisiae </i>using CRISPR-Cas systems. <i>Nucleic Acids Research </i>(2013); Jiang, W. et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems. <i>Nature Biotechnology </i>31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference).

0217The term “Cas9” or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A Cas9 nuclease is also referred to sometimes as a casn1 nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. <i>Science </i>337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an M1 strain of <i>Streptococcus pyogenes</i>.” Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. <i>Science </i>337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, <i>S. pyogenes </i>and <i>S. thermophilus</i>. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase.

0218A nuclease-inactivated Cas9 protein may interchangeably be referred to as a “dCas9” protein (for nuclease-“dead” Cas9). Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al., <i>Science. </i>337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28; 152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of <i>S. pyogenes </i>Cas9 (Jinek et al., <i>Science. </i>337:816-821(2012); Qi et al., Cell. 28; 152(5):1173-83 (2013)). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as “Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild type Cas9. In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.

0219In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length. In some embodiments, wild type Cas9 corresponds to Cas9 from <i>Streptococcus pyogenes </i>(NCBI Reference Sequence: NC_017053.1, SEQ ID NO: 1 (nucleotide); SEQ ID NO: 2 (amino acid)).

0220<tables id="TABLE-US-00002" num="00002"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 1)</entry></row><row><entry>ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGG</entry></row><row><entry></entry></row><row><entry>ATGGGCGGTGATCACTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGG</entry></row><row><entry></entry></row><row><entry>TTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCT</entry></row><row><entry></entry></row><row><entry>CTTTTATTTGGCAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGAC</entry></row><row><entry></entry></row><row><entry>AGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGG</entry></row><row><entry></entry></row><row><entry>AGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGA</entry></row><row><entry></entry></row><row><entry>CTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCC</entry></row><row><entry></entry></row><row><entry>TATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA</entry></row><row><entry></entry></row><row><entry>CTATCTATCATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGAT</entry></row><row><entry></entry></row><row><entry>TTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCA</entry></row><row><entry></entry></row><row><entry>TTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAAC</entry></row><row><entry></entry></row><row><entry>TATTTATCCAGTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCT</entry></row><row><entry></entry></row><row><entry>ATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAG</entry></row><row><entry></entry></row><row><entry>TAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGA</entry></row><row><entry></entry></row><row><entry>GAAATGGCTTGTTTGGGAATCTCATTGCTTTGTCATTGGGATTGACCCCT</entry></row><row><entry></entry></row><row><entry>AATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTC</entry></row><row><entry></entry></row><row><entry>AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAG</entry></row><row><entry></entry></row><row><entry>ATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATT</entry></row><row><entry></entry></row><row><entry>TTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCT</entry></row><row><entry></entry></row><row><entry>ATCAGCTTCAATGATTAAGCGCTACGATGAACATCATCAAGACTTGACTC</entry></row><row><entry></entry></row><row><entry>TTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATC</entry></row><row><entry></entry></row><row><entry>TTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGC</entry></row><row><entry></entry></row><row><entry>TAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGG</entry></row><row><entry></entry></row><row><entry>ATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGC</entry></row><row><entry></entry></row><row><entry>AAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGG</entry></row><row><entry></entry></row><row><entry>TGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAA</entry></row><row><entry></entry></row><row><entry>AAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTAT</entry></row><row><entry></entry></row><row><entry>TATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCG</entry></row><row><entry></entry></row><row><entry>GAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATA</entry></row><row><entry></entry></row><row><entry>AAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAA</entry></row><row><entry></entry></row><row><entry>AATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTA</entry></row><row><entry></entry></row><row><entry>TTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAA</entry></row><row><entry></entry></row><row><entry>TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGAT</entry></row><row><entry></entry></row><row><entry>TTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGA</entry></row><row><entry></entry></row><row><entry>TTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTG</entry></row><row><entry></entry></row><row><entry>AAGATAGATTTAATGCTTCATTAGGCGCCTACCATGATTTGCTAAAAATT</entry></row><row><entry></entry></row><row><entry>ATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGA</entry></row><row><entry></entry></row><row><entry>GGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGGGATGATTGAGG</entry></row><row><entry></entry></row><row><entry>AAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAG</entry></row><row><entry></entry></row><row><entry>CTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGAT</entry></row><row><entry></entry></row><row><entry>TAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGA</entry></row><row><entry></entry></row><row><entry>AATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGAT</entry></row><row><entry></entry></row><row><entry>AGTTTGACATTTAAAGAAGATATTCAAAAAGCACAGGTGTCTGGACAAGG</entry></row><row><entry></entry></row><row><entry>CCATAGTTTACATGAACAGATTGCTAACTTAGCTGGCAGTCCTGCTATTA</entry></row><row><entry></entry></row><row><entry>AAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACTGGTCAAAGTA</entry></row><row><entry></entry></row><row><entry>ATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCA</entry></row><row><entry></entry></row><row><entry>GACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCG</entry></row><row><entry></entry></row><row><entry>AAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTT</entry></row><row><entry></entry></row><row><entry>GAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTACAAAA</entry></row><row><entry></entry></row><row><entry>TGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTG</entry></row><row><entry></entry></row><row><entry>ATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCA</entry></row><row><entry></entry></row><row><entry>ATAGACAATAAGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGA</entry></row><row><entry></entry></row><row><entry>TAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGAC</entry></row><row><entry></entry></row><row><entry>AACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACG</entry></row><row><entry></entry></row><row><entry>AAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAA</entry></row><row><entry></entry></row><row><entry>ACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTT</entry></row><row><entry></entry></row><row><entry>TGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGA</entry></row><row><entry></entry></row><row><entry>GAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAA</entry></row><row><entry></entry></row><row><entry>AGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCC</entry></row><row><entry></entry></row><row><entry>ATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATAT</entry></row><row><entry></entry></row><row><entry>CCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGT</entry></row><row><entry></entry></row><row><entry>TCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAA</entry></row><row><entry></entry></row><row><entry>AATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACA</entry></row><row><entry></entry></row><row><entry>CTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGA</entry></row><row><entry></entry></row><row><entry>AACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCA</entry></row><row><entry></entry></row><row><entry>AAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAG</entry></row><row><entry></entry></row><row><entry>ACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAA</entry></row><row><entry></entry></row><row><entry>GCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTG</entry></row><row><entry></entry></row><row><entry>ATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAA</entry></row><row><entry></entry></row><row><entry>GGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAAT</entry></row><row><entry></entry></row><row><entry>TATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTA</entry></row><row><entry></entry></row><row><entry>AAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATAT</entry></row><row><entry></entry></row><row><entry>AGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGG</entry></row><row><entry></entry></row><row><entry>AGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATT</entry></row><row><entry></entry></row><row><entry>TTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGAT</entry></row><row><entry></entry></row><row><entry>AACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGA</entry></row><row><entry></entry></row><row><entry>GATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATG</entry></row><row><entry></entry></row><row><entry>CCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCA</entry></row><row><entry></entry></row><row><entry>ATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCT</entry></row><row><entry></entry></row><row><entry>TGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAAC</entry></row><row><entry></entry></row><row><entry>GATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCC</entry></row><row><entry></entry></row><row><entry>ATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGA</entry></row><row><entry></entry></row><row><entry>CTGA</entry></row><row><entry></entry></row><row><entry>(SEQ ID NO: 2)</entry></row><row><entry>MDKK<u style="double">YSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGA</u></entry></row><row><entry></entry></row><row><entry><u style="double">LLFGSGET</u>AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR</entry></row><row><entry></entry></row><row><entry>LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKAD</entry></row><row><entry></entry></row><row><entry>LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENP</entry></row><row><entry></entry></row><row><entry>INASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALSLGLTP</entry></row><row><entry></entry></row><row><entry>NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI</entry></row><row><entry></entry></row><row><entry>LLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI</entry></row><row><entry></entry></row><row><entry>FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR</entry></row><row><entry></entry></row><row><entry>KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY</entry></row><row><entry></entry></row><row><entry>YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK</entry></row><row><entry></entry></row><row><entry>NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD</entry></row><row><entry></entry></row><row><entry>LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKI</entry></row><row><entry></entry></row><row><entry>IKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQ</entry></row><row><entry></entry></row><row><entry>LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD</entry></row><row><entry></entry></row><row><entry>SLTFKEDIQKAQVSGQG<u style="double">HSLHEQIANLAGSPAIKKGILQTVKIVDELVKV</u></entry></row><row><entry></entry></row><row><entry><u style="double">MGHKPENIVIEMAR</u>ENQTTQK<u style="single">GQKNSRERMKRIEEGIKELGSQILKEHPV</u></entry></row><row><entry></entry></row><row><entry><u style="single">ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDS</u></entry></row><row><entry></entry></row><row><entry><u style="single">IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT</u></entry></row><row><entry></entry></row><row><entry><u style="single">K</u>AERG<u style="double">GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR</u></entry></row><row><entry></entry></row><row><entry><u style="double">EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY</u></entry></row><row><entry></entry></row><row><entry><u style="double">PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT</u></entry></row><row><entry></entry></row><row><entry><u style="double">LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ</u></entry></row><row><entry></entry></row><row><entry><u style="double">T</u>GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK</entry></row><row><entry></entry></row><row><entry>GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY</entry></row><row><entry></entry></row><row><entry>SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED</entry></row><row><entry></entry></row><row><entry>NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP</entry></row><row><entry></entry></row><row><entry>IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS</entry></row><row><entry></entry></row><row><entry>ITGLYETRIDLSQLGGD</entry></row><row><entry></entry></row><row><entry>(single underline: HNH domain; double underline:</entry></row><row><entry>RuvC domain)</entry></row></tbody></tgroup></table></tables>

0221In some embodiments, wild type Cas9 corresponds to, or comprises SEQ ID NO:3 (nucleotide) and/or SEQ ID NO: 4 (amino acid):

0222<tables id="TABLE-US-00003" num="00003"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 3)</entry></row><row><entry>ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGG</entry></row><row><entry></entry></row><row><entry>ATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGG</entry></row><row><entry></entry></row><row><entry>TGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCC</entry></row><row><entry></entry></row><row><entry>CTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAAC</entry></row><row><entry></entry></row><row><entry>CGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAG</entry></row><row><entry></entry></row><row><entry>AAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGT</entry></row><row><entry></entry></row><row><entry>TTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCC</entry></row><row><entry></entry></row><row><entry>CATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAA</entry></row><row><entry></entry></row><row><entry>CGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGAC</entry></row><row><entry></entry></row><row><entry>CTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCA</entry></row><row><entry></entry></row><row><entry>CTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAAC</entry></row><row><entry></entry></row><row><entry>TGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCT</entry></row><row><entry></entry></row><row><entry>ATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTC</entry></row><row><entry></entry></row><row><entry>TAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGA</entry></row><row><entry></entry></row><row><entry>AAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCA</entry></row><row><entry></entry></row><row><entry>AATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAG</entry></row><row><entry></entry></row><row><entry>TAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAG</entry></row><row><entry></entry></row><row><entry>ATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATC</entry></row><row><entry></entry></row><row><entry>CTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTT</entry></row><row><entry></entry></row><row><entry>ATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACAC</entry></row><row><entry></entry></row><row><entry>TTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATA</entry></row><row><entry></entry></row><row><entry>TTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGC</entry></row><row><entry></entry></row><row><entry>GAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGG</entry></row><row><entry></entry></row><row><entry>ATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGA</entry></row><row><entry></entry></row><row><entry>AAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGG</entry></row><row><entry></entry></row><row><entry>CGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCA</entry></row><row><entry></entry></row><row><entry>AAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTAC</entry></row><row><entry></entry></row><row><entry>TATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAG</entry></row><row><entry></entry></row><row><entry>AAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATA</entry></row><row><entry></entry></row><row><entry>AAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAG</entry></row><row><entry></entry></row><row><entry>AATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTA</entry></row><row><entry></entry></row><row><entry>TTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCA</entry></row><row><entry></entry></row><row><entry>TGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGAT</entry></row><row><entry></entry></row><row><entry>CTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGA</entry></row><row><entry></entry></row><row><entry>CTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAG</entry></row><row><entry></entry></row><row><entry>AAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATA</entry></row><row><entry></entry></row><row><entry>ATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGA</entry></row><row><entry></entry></row><row><entry>AGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGG</entry></row><row><entry></entry></row><row><entry>AAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAG</entry></row><row><entry></entry></row><row><entry>TTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTAT</entry></row><row><entry></entry></row><row><entry>CAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAA</entry></row><row><entry></entry></row><row><entry>AGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGAC</entry></row><row><entry></entry></row><row><entry>TCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGG</entry></row><row><entry></entry></row><row><entry>GGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCA</entry></row><row><entry></entry></row><row><entry>AAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTC</entry></row><row><entry></entry></row><row><entry>ATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAA</entry></row><row><entry></entry></row><row><entry>TCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAA</entry></row><row><entry></entry></row><row><entry>TAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCT</entry></row><row><entry></entry></row><row><entry>GTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACA</entry></row><row><entry></entry></row><row><entry>AAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTAT</entry></row><row><entry></entry></row><row><entry>CTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGAT</entry></row><row><entry></entry></row><row><entry>TCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAG</entry></row><row><entry></entry></row><row><entry>TGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGC</entry></row><row><entry></entry></row><row><entry>GGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTA</entry></row><row><entry></entry></row><row><entry>ACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTAT</entry></row><row><entry></entry></row><row><entry>TAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGA</entry></row><row><entry></entry></row><row><entry>TACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATT</entry></row><row><entry></entry></row><row><entry>CGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAG</entry></row><row><entry></entry></row><row><entry>AAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATG</entry></row><row><entry></entry></row><row><entry>CGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAA</entry></row><row><entry></entry></row><row><entry>TACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGA</entry></row><row><entry></entry></row><row><entry>CGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAG</entry></row><row><entry></entry></row><row><entry>CCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATC</entry></row><row><entry></entry></row><row><entry>ACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGG</entry></row><row><entry></entry></row><row><entry>GGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGA</entry></row><row><entry></entry></row><row><entry>GAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTG</entry></row><row><entry></entry></row><row><entry>CAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGA</entry></row><row><entry></entry></row><row><entry>TAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCT</entry></row><row><entry></entry></row><row><entry>TCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAG</entry></row><row><entry></entry></row><row><entry>AAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAAC</entry></row><row><entry></entry></row><row><entry>GATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGG</entry></row><row><entry></entry></row><row><entry>CGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAG</entry></row><row><entry></entry></row><row><entry>TATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGC</entry></row><row><entry></entry></row><row><entry>CGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGA</entry></row><row><entry></entry></row><row><entry>ATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAA</entry></row><row><entry></entry></row><row><entry>GATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGA</entry></row><row><entry></entry></row><row><entry>CGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTG</entry></row><row><entry></entry></row><row><entry>ATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAA</entry></row><row><entry></entry></row><row><entry>CCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAA</entry></row><row><entry></entry></row><row><entry>CCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCA</entry></row><row><entry></entry></row><row><entry>AACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAA</entry></row><row><entry></entry></row><row><entry>TCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGG</entry></row><row><entry></entry></row><row><entry>TGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACC</entry></row><row><entry></entry></row><row><entry>ATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGAC</entry></row><row><entry></entry></row><row><entry>AAGGCTGCAGGA</entry></row><row><entry></entry></row><row><entry>(SEQ ID NO: 4)</entry></row><row><entry>MDKK<u style="double">YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA</u></entry></row><row><entry></entry></row><row><entry><u style="double">LLFDSGET</u>AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR</entry></row><row><entry></entry></row><row><entry>LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD</entry></row><row><entry></entry></row><row><entry>LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP</entry></row><row><entry></entry></row><row><entry>INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP</entry></row><row><entry></entry></row><row><entry>NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI</entry></row><row><entry></entry></row><row><entry>LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI</entry></row><row><entry></entry></row><row><entry>FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR</entry></row><row><entry></entry></row><row><entry>KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY</entry></row><row><entry></entry></row><row><entry>YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK</entry></row><row><entry></entry></row><row><entry>NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD</entry></row><row><entry></entry></row><row><entry>LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI</entry></row><row><entry></entry></row><row><entry>IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ</entry></row><row><entry></entry></row><row><entry>LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD</entry></row><row><entry></entry></row><row><entry>SLTFKEDIQKAQVSGQG<u style="double">DSLHEHIANLAGSPAIKKGILQTVKVVDELVKV</u></entry></row><row><entry></entry></row><row><entry><u style="double">MGRHKPENIVIEMA</u>RENQTTQK<u style="single">GQKNSRERMKRIEEGIKELGSQILKEHP</u></entry></row><row><entry></entry></row><row><entry><u style="single">VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD</u></entry></row><row><entry></entry></row><row><entry><u style="single">SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL</u></entry></row><row><entry></entry></row><row><entry><u style="single">TK</u>AERG<u style="double">GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI</u></entry></row><row><entry></entry></row><row><entry><u style="double">REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK</u></entry></row><row><entry></entry></row><row><entry><u style="double">YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI</u></entry></row><row><entry></entry></row><row><entry><u style="double">TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV</u></entry></row><row><entry></entry></row><row><entry><u style="double">QT</u>GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE</entry></row><row><entry></entry></row><row><entry>KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK</entry></row><row><entry></entry></row><row><entry>YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE</entry></row><row><entry></entry></row><row><entry>DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK</entry></row><row><entry></entry></row><row><entry>PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ</entry></row><row><entry></entry></row><row><entry>SITGLYETRIDLSQLGGD</entry></row><row><entry></entry></row><row><entry>(single underline: HNH domain; double underline:</entry></row><row><entry>RuvC domain)</entry></row></tbody></tgroup></table></tables>

0223In some embodiments, wild type Cas9 corresponds to Cas9 from <i>Streptococcus pyogenes </i>(NCBI Reference Sequence: NC_002737.2, SEQ ID NO: 5 (nucleotide); and Uniport Reference Sequence: Q99ZW2, SEQ ID NO: 6 (amino acid).

0224<tables id="TABLE-US-00004" num="00004"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 5)</entry></row><row><entry>ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGG</entry></row><row><entry></entry></row><row><entry>ATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGG</entry></row><row><entry></entry></row><row><entry>TTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCT</entry></row><row><entry></entry></row><row><entry>CTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGAC</entry></row><row><entry></entry></row><row><entry>AGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGG</entry></row><row><entry></entry></row><row><entry>AGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGA</entry></row><row><entry></entry></row><row><entry>CTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCC</entry></row><row><entry></entry></row><row><entry>TATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA</entry></row><row><entry></entry></row><row><entry>CTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGAT</entry></row><row><entry></entry></row><row><entry>TTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCA</entry></row><row><entry></entry></row><row><entry>TTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAAC</entry></row><row><entry></entry></row><row><entry>TATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCT</entry></row><row><entry></entry></row><row><entry>ATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAG</entry></row><row><entry></entry></row><row><entry>TAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGA</entry></row><row><entry></entry></row><row><entry>AAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCT</entry></row><row><entry></entry></row><row><entry>AATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTC</entry></row><row><entry></entry></row><row><entry>AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAG</entry></row><row><entry></entry></row><row><entry>ATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATT</entry></row><row><entry></entry></row><row><entry>TTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCT</entry></row><row><entry></entry></row><row><entry>ATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTC</entry></row><row><entry></entry></row><row><entry>TTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATC</entry></row><row><entry></entry></row><row><entry>TTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGC</entry></row><row><entry></entry></row><row><entry>TAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGG</entry></row><row><entry></entry></row><row><entry>ATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGC</entry></row><row><entry></entry></row><row><entry>AAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGG</entry></row><row><entry></entry></row><row><entry>TGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAA</entry></row><row><entry></entry></row><row><entry>AAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTAT</entry></row><row><entry></entry></row><row><entry>TATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCG</entry></row><row><entry></entry></row><row><entry>GAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATA</entry></row><row><entry></entry></row><row><entry>AAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAA</entry></row><row><entry></entry></row><row><entry>AATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTA</entry></row><row><entry></entry></row><row><entry>TTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAA</entry></row><row><entry></entry></row><row><entry>TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGAT</entry></row><row><entry></entry></row><row><entry>TTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGA</entry></row><row><entry></entry></row><row><entry>TTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTG</entry></row><row><entry></entry></row><row><entry>AAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATT</entry></row><row><entry></entry></row><row><entry>ATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGA</entry></row><row><entry></entry></row><row><entry>GGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGG</entry></row><row><entry></entry></row><row><entry>AAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAG</entry></row><row><entry></entry></row><row><entry>CTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGAT</entry></row><row><entry></entry></row><row><entry>TAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGA</entry></row><row><entry></entry></row><row><entry>AATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGAT</entry></row><row><entry></entry></row><row><entry>AGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGG</entry></row><row><entry></entry></row><row><entry>CGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTA</entry></row><row><entry></entry></row><row><entry>AAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTA</entry></row><row><entry></entry></row><row><entry>ATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAA</entry></row><row><entry></entry></row><row><entry>TCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAA</entry></row><row><entry></entry></row><row><entry>TCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCT</entry></row><row><entry></entry></row><row><entry>GTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCA</entry></row><row><entry></entry></row><row><entry>AAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAA</entry></row><row><entry></entry></row><row><entry>GTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGAT</entry></row><row><entry></entry></row><row><entry>TCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATC</entry></row><row><entry></entry></row><row><entry>GGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGA</entry></row><row><entry></entry></row><row><entry>GACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTA</entry></row><row><entry></entry></row><row><entry>ACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTAT</entry></row><row><entry></entry></row><row><entry>CAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAA</entry></row><row><entry></entry></row><row><entry>TTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATT</entry></row><row><entry></entry></row><row><entry>CGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCG</entry></row><row><entry></entry></row><row><entry>AAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATG</entry></row><row><entry></entry></row><row><entry>CCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAA</entry></row><row><entry></entry></row><row><entry>TATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGA</entry></row><row><entry></entry></row><row><entry>TGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCG</entry></row><row><entry></entry></row><row><entry>CAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATT</entry></row><row><entry></entry></row><row><entry>ACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGG</entry></row><row><entry></entry></row><row><entry>GGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGC</entry></row><row><entry></entry></row><row><entry>GCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTA</entry></row><row><entry></entry></row><row><entry>CAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGA</entry></row><row><entry></entry></row><row><entry>CAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTT</entry></row><row><entry></entry></row><row><entry>TTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAA</entry></row><row><entry></entry></row><row><entry>AAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCAC</entry></row><row><entry></entry></row><row><entry>AATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAG</entry></row><row><entry></entry></row><row><entry>CTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAA</entry></row><row><entry></entry></row><row><entry>TATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGC</entry></row><row><entry></entry></row><row><entry>CGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGA</entry></row><row><entry></entry></row><row><entry>ATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAA</entry></row><row><entry></entry></row><row><entry>GATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGA</entry></row><row><entry></entry></row><row><entry>TGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAG</entry></row><row><entry></entry></row><row><entry>ATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAA</entry></row><row><entry></entry></row><row><entry>CCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAA</entry></row><row><entry></entry></row><row><entry>TCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTA</entry></row><row><entry></entry></row><row><entry>AACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAA</entry></row><row><entry></entry></row><row><entry>TCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGG</entry></row><row><entry></entry></row><row><entry>TGACTGA</entry></row><row><entry></entry></row><row><entry>(SEQ ID NO: 6)</entry></row><row><entry>MDKK<u style="double">YSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA</u></entry></row><row><entry></entry></row><row><entry><u style="double">LLFDSGET</u>AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR</entry></row><row><entry></entry></row><row><entry>LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD</entry></row><row><entry></entry></row><row><entry>LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP</entry></row><row><entry></entry></row><row><entry>INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP</entry></row><row><entry></entry></row><row><entry>NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI</entry></row><row><entry></entry></row><row><entry>LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI</entry></row><row><entry></entry></row><row><entry>FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR</entry></row><row><entry></entry></row><row><entry>KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY</entry></row><row><entry></entry></row><row><entry>YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK</entry></row><row><entry></entry></row><row><entry>NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD</entry></row><row><entry></entry></row><row><entry>LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI</entry></row><row><entry></entry></row><row><entry>IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ</entry></row><row><entry></entry></row><row><entry>LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD</entry></row><row><entry></entry></row><row><entry>SLTFKEDIQKAQVSGQG<u style="double">DSLHEHIANLAGSPAIKKGILQTVKVVDELVKV</u></entry></row><row><entry></entry></row><row><entry><u style="double">MGRHKPENIVIEMA</u>RENQTTQK<u style="single">GQKNSRERMKRIEEGIKELGSQILKEHP</u></entry></row><row><entry></entry></row><row><entry><u style="single">VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD</u></entry></row><row><entry></entry></row><row><entry><u style="single">SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL</u></entry></row><row><entry></entry></row><row><entry><u style="single">TK</u>AERG<u style="double">GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI</u></entry></row><row><entry></entry></row><row><entry><u style="double">REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK</u></entry></row><row><entry></entry></row><row><entry><u style="double">YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI</u></entry></row><row><entry></entry></row><row><entry><u style="double">TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV</u></entry></row><row><entry></entry></row><row><entry><u style="double">QT</u>GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE</entry></row><row><entry></entry></row><row><entry>KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK</entry></row><row><entry></entry></row><row><entry>YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE</entry></row><row><entry></entry></row><row><entry>DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK</entry></row><row><entry></entry></row><row><entry>PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ</entry></row><row><entry></entry></row><row><entry>SITGLYETRIDLSQLGGD</entry></row><row><entry></entry></row><row><entry>(single underline: HNH domain; double underline:</entry></row><row><entry>RuvC domain)</entry></row></tbody></tgroup></table></tables>

0225In some embodiments, Cas9 refers to Cas9 from: <i>Corynebacterium ulcerans </i>(NCBI Refs: NC_015683.1, NC_017317.1); <i>Corynebacterium diphtheria </i>(NCBI Refs: NC_016782.1, NC_016786.1); <i>Spiroplasma syrphidicola </i>(NCBI Ref: NC_021284.1); <i>Prevotella intermedia </i>(NCBI Ref: NC_017861.1); <i>Spiroplasma taiwanense </i>(NCBI Ref: NC_021846.1); <i>Streptococcus iniae </i>(NCBI Ref: NC_021314.1); <i>Belliella baltica </i>(NCBI Ref: NC_018010.1); <i>Psychroflexus torquisl </i>(NCBI Ref: NC_018721.1); <i>Streptococcus thermophilus </i>(NCBI Ref: YP_820832.1), <i>Listeria innocua </i>(NCBI Ref: NP_472073.1), <i>Campylobacter jejuni </i>(NCBI Ref: YP_002344900.1) or <i>Neisseria. meningitidis </i>(NCBI Ref: YP_002342100.1) or to a Cas9 from any of the organisms listed in Example 5.

0226In some embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. For example, in some embodiments, a dCas9 domain comprises D10A and/or H840A mutation.

0227<tables id="TABLE-US-00005" num="00005"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>dCas9 (D10A and H840A):</entry></row><row><entry>(SEQ ID NO: 7)</entry></row><row><entry>MDKK<u style="single"><b>YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA</b></u></entry></row><row><entry></entry></row><row><entry><u style="single"><b>LLFDSGET</b></u>AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR</entry></row><row><entry></entry></row><row><entry>LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD</entry></row><row><entry></entry></row><row><entry>LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP</entry></row><row><entry></entry></row><row><entry>INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP</entry></row><row><entry></entry></row><row><entry>NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI</entry></row><row><entry></entry></row><row><entry>LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI</entry></row><row><entry></entry></row><row><entry>FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR</entry></row><row><entry></entry></row><row><entry>KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY</entry></row><row><entry></entry></row><row><entry>YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK</entry></row><row><entry></entry></row><row><entry>NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD</entry></row><row><entry></entry></row><row><entry>LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI</entry></row><row><entry></entry></row><row><entry>IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ</entry></row><row><entry></entry></row><row><entry>LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD</entry></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00001" num="00001"><img file="US11268082B2_D0001.tif" /></chemistry></entry></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00002" num="00002"><img file="US11268082B2_D0002.tif" /></chemistry></entry></row><row><entry></entry></row><row><entry><u style="single"><b>PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKD</b></u></entry></row><row><entry></entry></row><row><entry><u style="single"><b>DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN</b></u></entry></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00003" num="00003"><img file="US11268082B2_D0003.tif" /></chemistry></entry></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00004" num="00004"><img file="US11268082B2_D0004.tif" /></chemistry></entry></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00005" num="00005"><img file="US11268082B2_D0005.tif" /></chemistry></entry></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00006" num="00006"><img file="US11268082B2_D0006.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00007" num="00007"><img file="US11268082B2_D0007.tif" /></chemistry></entry></row><row><entry></entry></row><row><entry>EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLP</entry></row><row><entry></entry></row><row><entry>KYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP</entry></row><row><entry></entry></row><row><entry>EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD</entry></row><row><entry></entry></row><row><entry>KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH</entry></row><row><entry></entry></row><row><entry>QSITGLYETRIDLSQLGGD (single underline: HNH domain;</entry></row><row><entry></entry></row><row><entry>(double underline: RuvC domain).</entry></row></tbody></tgroup></table></tables>

0228In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequence provided in SEQ ID NO: 6, or at corresponding positions in any of the amino acid sequences provided in another Cas9 domain, such as any of the Cas9 proteins provided herein. Without wishing to be bound by any particular theory, the presence of the catalytic residue H840 restores the activity of the Cas9 to cleave the non-edited (e.g., non-deaminated) strand containing a G opposite the targeted C. Restoration of H840 (e.g., from A840) does not result in the cleavage of the target strand containing the C. Such Cas9 variants are able to generate a single-strand DNA break (nick) at a specific location based on the gRNA-defined target sequence, leading to repair of the non-edited strand, ultimately resulting in a G to A change on the non-edited strand. A schematic representation of this process is shown in <figref idref="DRAWINGS">FIG. 108</figref>. Briefly, the C of a C-G basepair can be deaminated to a U by a deaminase, e.g., an APOBEC deamonase. Nicking the non-edited strand, having the G, facilitates removal of the G via mismatch repair mechanisms. UGI inhibits UDG, which prevents removal of the U.

0229In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which, e.g., result in nuclease inactivated Cas9 (dCas9). Such mutations, by way of example, include other amino acid substitutions at D10 and H820, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain). In some embodiments, variants or homologues of dCas9 (e.g., variants of SEQ ID NO: 6) are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to SEQ ID NO: 6. In some embodiments, variants of dCas9 (e.g., variants of SEQ ID NO: 6) are provided having amino acid sequences which are shorter, or longer than SEQ ID NO: 6, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.

0230In some embodiments, Cas9 fusion proteins as provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas9 sequence, but only a fragment thereof. For example, in some embodiments, a Cas9 fusion protein provided herein comprises a Cas9 fragment, wherein the fragment binds crRNA and tracrRNA or sgRNA, but does not comprise a functional nuclease domain, e.g., in that it comprises only a truncated version of a nuclease domain or no nuclease domain at all. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those of skill in the art.

0231In some embodiments, Cas9 refers to Cas9 from: <i>Corynebacterium ulcerans </i>(NCBI Refs: NC_015683.1, NC_017317.1); <i>Corynebacterium diphtheria </i>(NCBI Refs: NC_016782.1, NC_016786.1); <i>Spiroplasma syrphidicola </i>(NCBI Ref: NC_021284.1); <i>Prevotella intermedia </i>(NCBI Ref: NC_017861.1); <i>Spiroplasma taiwanense </i>(NCBI Ref: NC_021846.1); <i>Streptococcus iniae </i>(NCBI Ref: NC_021314.1); <i>Belliella baltica </i>(NCBI Ref: NC_018010.1); <i>Psychroflexus torquis </i>I (NCBI Ref: NC_018721.1); <i>Streptococcus thermophilus </i>(NCBI Ref: YP_820832.1); <i>Listeria innocua </i>(NCBI Ref: NP_472073.1); <i>Campylobacter jejuni </i>(NCBI Ref: YP_002344900.1); or <i>Neisseria. meningitidis </i>(NCBI Ref: YP_002342100.1).

0232The term “deaminase” or “deaminase domain,” as used herein, refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase, catalyzing the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase domain, catalyzing the hydrolytic deamination of cytosine to uracil. In some embodiments, the deaminase or deaminase domain is a naturally-occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is a variant of a naturally-occurring deaminase from an organism, that does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase from an organism.

0233The term “effective amount,” as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a nuclease may refer to the amount of the nuclease that is sufficient to induce cleavage of a target site specifically bound and cleaved by the nuclease. In some embodiments, an effective amount of a fusion protein provided herein, e.g., of a fusion protein comprising a nuclease-inactive Cas9 domain and a nucleic acid editing domain (e.g., a deaminase domain) may refer to the amount of the fusion protein that is sufficient to induce editing of a target site specifically bound and edited by the fusion protein. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a fusion protein, a nuclease, a deaminase, a recombinase, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific allele, genome, or target site to be edited, on the cell or tissue being targeted, and on the agent being used.

0234The term “linker,” as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., two domains of a fusion protein, such as, for example, a nuclease-inactive Cas9 domain and a nucleic acid editing domain (e.g., a deaminase domain). A linker may be, for example, an amino acid sequence, a peptide, or a polymer of any length and composition. In some embodiments, a linker joins a gRNA binding domain of an RNA-programmable nuclease, including a Cas9 nuclease domain, and the catalytic domain of a nucleic-acid editing protein. In some embodiments, a linker joins a dCas9 and a nucleic-acid editing protein. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 1-100 amino acids in length, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

0235The term “mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, <i>Molecular Cloning: A Laboratory Manual </i>(4^thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)).

0236The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g. nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

0237The term “nucleic acid editing domain,” as used herein refers to a protein or enzyme capable of making one or more modifications (e.g., deamination of a cytidine residue) to a nucleic acid (e.g., DNA or RNA). Exemplary nucleic acid editing domains include, but are not limited to a deaminase, a nuclease, a nickase, a recombinase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain. In some embodiments the nucleic acid editing domain is a deaminase (e.g., a cytidine deaminase, such as an APOBEC or an AID deaminase).

0238The term “proliferative disease,” as used herein, refers to any disease in which cell or tissue homeostasis is disturbed in that a cell or cell population exhibits an abnormally elevated proliferation rate. Proliferative diseases include hyperproliferative diseases, such as pre-neoplastic hyperplastic conditions and neoplastic diseases. Neoplastic diseases are characterized by an abnormal proliferation of cells and include both benign and malignant neoplasias. Malignant neoplasia is also referred to as cancer.

0239The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a nucleic-acid editing protein. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent. In some embodiments, a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, <i>Molecular Cloning: A Laboratory Manual </i>(4^thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

0240The term “subject,” as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development.

0241The term “target site” refers to a sequence within a nucleic acid molecule that is deaminated by a deaminase or a fusion protein comprising a deaminase, (e.g., a dCas9-deaminase fusion protein provided herein).

0242The terms “treatment,” “treat,” and “treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. As used herein, the terms “treatment,” “treat,” and “treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

0243The term “recombinant” as used herein in the context of proteins or nucleic acids refers to proteins or nucleic acids that do not occur in nature, but are the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations as compared to any naturally occurring sequence.

0244The term “pharmaceutical composition,” as used herein, refers to a composition that can be administrated to a subject in the context of treatment of a disease or disorder. In some embodiments, a pharmaceutical composition comprises an active ingredient, e.g., a nuclease or a nucleic acid encoding a nuclease, and a pharmaceutically acceptable excipient.

0245The term “base editor (BE),” or “nucleobase editor (NBE),” as used herein, refers to an agent comprising a polypeptide that is capable of making a modification to a base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the base editor is capable of deaminating a base within a nucleic acid. In some embodiments, the base editor is capable of deaminating a base within a DNA molecule. In some embodiments, the base editor is capable of deaminating an cytosine (C) in DNA. In some embodiments, the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) fused to a cytidine deaminase domain. In some embodiments, the base editor comprises a Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein fused to a cytidine deaminase. In some embodiments, the base editor comprises a Cas9 nickase (nCas9) fused to an cytidine deaminase. In some embodiments, the base editor comprises a nuclease-inactive Cas9 (dCas9) fused to a cytidine deaminase. In some embodiments, the base editor is fused to an inhibitor of base excision repair, for example, a UGI domain. In some embodiments, the base editor comprises a CasX protein fused to a cytidine deaminase. In some embodiments, the base editor comprises a CasY protein fused to a cytidine deaminase. In some embodiments, the base editor comprises a Cpf1 protein fused to a cytidine deaminase. In some embodiments, the base editor comprises a C2c1 protein fused to a cytidine deaminase. In some embodiments, the base editor comprises a C2c2 protein fused to a cytidine deaminase. In some embodiments, the base editor comprises a C2c3 protein fused to a cytidine deaminase. In some embodiments, the base editor comprises an Argonaute protein fused to a cytidine deaminase.

0246The term “uracil glycosylase inhibitor” or “UGI,” as used herein, refers to a protein that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme.

0247The term “Cas9 nickase,” as used herein, refers to a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule (e.g., a duplexed DNA molecule). In some embodiments, a Cas9 nickase comprises a D10A mutation and has a histidine at position H840 of SEQ ID NO: 6, or a corresponding mutation in another Cas9 domain, such as any of the Cas9 proteins provided herein. For example, a Cas9 nickase may comprise the amino acid sequence as set forth in SEQ ID NO: 8. Such a Cas9 nickase has an active HNH nuclease domain and is able to cleave the non-targeted strand of DNA, i.e., the strand bound by the gRNA. Further, such a Cas9 nickase has an inactive RuvC nuclease domain and is not able to cleave the targeted strand of the DNA, i.e., the strand where base editing is desired.

0248Exemplary Cas9 nickase (Cloning vector pPlatTET-gRNA2; Accession No. BAV54124).

0249<tables id="TABLE-US-00006" num="00006"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 8)</entry></row><row><entry>MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA</entry></row><row><entry></entry></row><row><entry>LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR</entry></row><row><entry></entry></row><row><entry>LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD</entry></row><row><entry></entry></row><row><entry>LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP</entry></row><row><entry></entry></row><row><entry>INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP</entry></row><row><entry></entry></row><row><entry>NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI</entry></row><row><entry></entry></row><row><entry>LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI</entry></row><row><entry></entry></row><row><entry>FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR</entry></row><row><entry></entry></row><row><entry>KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY</entry></row><row><entry></entry></row><row><entry>YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK</entry></row><row><entry></entry></row><row><entry>NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD</entry></row><row><entry></entry></row><row><entry>LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI</entry></row><row><entry></entry></row><row><entry>IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ</entry></row><row><entry></entry></row><row><entry>LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD</entry></row><row><entry></entry></row><row><entry>SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV</entry></row><row><entry></entry></row><row><entry>MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP</entry></row><row><entry></entry></row><row><entry>VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD</entry></row><row><entry></entry></row><row><entry>SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL</entry></row><row><entry></entry></row><row><entry>TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI</entry></row><row><entry></entry></row><row><entry>REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK</entry></row><row><entry></entry></row><row><entry>YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI</entry></row><row><entry></entry></row><row><entry>TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV</entry></row><row><entry></entry></row><row><entry>QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE</entry></row><row><entry></entry></row><row><entry>KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK</entry></row><row><entry></entry></row><row><entry>YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE</entry></row><row><entry></entry></row><row><entry>DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK</entry></row><row><entry></entry></row><row><entry>PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ</entry></row><row><entry></entry></row><row><entry>SITGLYETRIDLSQLGGD</entry></row></tbody></tgroup></table></tables>

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

0250Some aspects of this disclosure provide fusion proteins that comprise a domain capable of binding to a nucleotide sequence (e.g., a Cas9, or a Cpf1 protein) and an enzyme domain, for example, a DNA-editing domain, such as, e.g., a deaminase domain. The deamination of a nucleobase by a deaminase can lead to a point mutation at the respective residue, which is referred to herein as nucleic acid editing. Fusion proteins comprising a Cas9 variant or domain and a DNA editing domain can thus be used for the targeted editing of nucleic acid sequences. Such fusion proteins are useful for targeted editing of DNA in vitro, e.g., for the generation of mutant cells or animals; for the introduction of targeted mutations, e.g., for the correction of genetic defects in cells ex vivo, e.g., in cells obtained from a subject that are subsequently re-introduced into the same or another subject; and for the introduction of targeted mutations, e.g., the correction of genetic defects or the introduction of deactivating mutations in disease-associated genes in a subject. Typically, the Cas9 domain of the fusion proteins described herein does not have any nuclease activity but instead is a Cas9 fragment or a dCas9 protein or domain. Other aspects of the invention provide fusion proteins that comprise (i) a domain capable of binding to a nucleic acid sequence (e.g., a Cas9, or a Cpf1 protein); (ii) an enzyme domain, for example, a DNA-editing domain (e.g., a deaminase domain); and (iii) one or more uracil glycosylase inhibitor (UGI) domains. The presence of at least one UGI domain increases base editing efficiency compared to fusion proteins without a UGI domain. A fusion protein comprising two UGI domains further increases base editing efficiency and product purity compared to fusion proteins with one UGI domain or without a UGI domain. Methods for the use of Cas9 fusion proteins as described herein are also provided.

0000Nucleic Acid Programmable DNA Binding Proteins

0251Some aspects of the disclosure provide nucleic acid programmable DNA binding proteins, which may be used to guide a protein, such as a base editor, to a specific nucleic acid (e.g., DNA or RNA) sequence. It should be appreciated that any of the fusion proteins (e.g., base editors) provided herein may include any nucleic acid programmable DNA binding protein (napDNAbp). For example, any of the fusion proteins described herein that include a Cas9 domain, can use another napDNAbp, such as CasX, CasY, Cpf1, C2c1, C2c2, C2c3, and Argonaute, in place of the Cas9 domain. Nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute. One example of a nucleic acid programmable DNA-binding protein that has a different PAM specificity than Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats from <i>Prevotella </i>and <i>Francisella </i>1 (Cpf1). Similar to Cas9, Cpf1 is also a class 2 CRISPR effector. It has been shown that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, two enzymes from <i>Acidaminococcus </i>and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpf1 proteins are known in the art and have been described previously, for example, Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” <i>Cell </i>(165) 2016, p. 949-962; the entire contents of which are incorporated herein by reference.

0252Also useful in the present compositions and methods are nuclease-inactive Cpf1 (dCpf1) variants that may be used as a guide nucleotide sequence-programmable DNA-binding protein domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alpha-helical recognition lobe of Cas9. It was shown in Zetsche et al., <i>Cell, </i>163, 759-771, 2015 (which is incorporated herein by reference) that, the RuvC-like domain of Cpf1 is responsible for cleaving both DNA strands and inactivation of the RuvC-like domain inactivates Cpf1 nuclease activity. For example, mutations corresponding to D917A, E1006A, or D1255A in <i>Francisella novicida </i>Cpf1 (SEQ ID NO: 15) inactivate Cpf1 nuclease activity. In some embodiments, the dead Cpf1 (dCpf1) comprises mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/E1006A/D1255A in SEQ ID NO: 9. It is to be understood that any mutations, e.g., substitution mutations, deletions, or insertions, that inactivate the RuvC domain of Cpf1, may be used in accordance with the present disclosure.

0253In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein is a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In some embodiments, the Cpf1 protein is a nuclease inactive Cpf1 (dCpf1). In some embodiments, the Cpf1, the nCpf1, or the dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 9-24. In some embodiments, the dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 9-16, and comprises mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/E1006A/D1255A in SEQ ID NO: 9. In some embodiments, the dCpf1 protein comprises an amino acid sequence of any one SEQ ID NOs: 9-16. It should be appreciated that Cpf1 from other species may also be used in accordance with the present disclosure.

0000Wild type <i>Francisella novicida </i>Cpf1 (SEQ ID NO: 9) (D917, E1006, and D1255 are bolded and underlined)

0254<tables id="TABLE-US-00007" num="00007"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 9)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKA</entry></row><row><entry></entry></row><row><entry>KQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKS</entry></row><row><entry></entry></row><row><entry>AKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGI</entry></row><row><entry></entry></row><row><entry>ELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSII</entry></row><row><entry></entry></row><row><entry>YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKT</entry></row><row><entry></entry></row><row><entry>SEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI</entry></row><row><entry></entry></row><row><entry>NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT</entry></row><row><entry></entry></row><row><entry>TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT</entry></row><row><entry></entry></row><row><entry>DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKY</entry></row><row><entry></entry></row><row><entry>LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLA</entry></row><row><entry></entry></row><row><entry>QISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSED</entry></row><row><entry></entry></row><row><entry>KANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF</entry></row><row><entry></entry></row><row><entry>ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENK</entry></row><row><entry></entry></row><row><entry>GEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN</entry></row><row><entry></entry></row><row><entry>GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSI</entry></row><row><entry></entry></row><row><entry>DEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR</entry></row><row><entry></entry></row><row><entry>PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIA</entry></row><row><entry></entry></row><row><entry>NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEI</entry></row><row><entry></entry></row><row><entry>NLLLKEKANDVHILSI<u style="single"><b>D</b></u>RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMK</entry></row><row><entry></entry></row><row><entry>TNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYN</entry></row><row><entry></entry></row><row><entry>AIVVF<u style="single"><b>E</b></u>DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG</entry></row><row><entry></entry></row><row><entry>VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYE</entry></row><row><entry></entry></row><row><entry>SVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR</entry></row><row><entry></entry></row><row><entry>LINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD</entry></row><row><entry></entry></row><row><entry>KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNM</entry></row><row><entry></entry></row><row><entry>PQDA<u style="single"><b>D</b></u>ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN</entry></row></tbody></tgroup></table></tables>

0255<i>Francisella novicida </i>Cpf1 D917A (SEQ ID NO: 10) (A917, E1006, and D1255 are bolded and underlined)

0256<tables id="TABLE-US-00008" num="00008"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 10)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKA</entry></row><row><entry></entry></row><row><entry>KQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKS</entry></row><row><entry></entry></row><row><entry>AKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGI</entry></row><row><entry></entry></row><row><entry>ELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSII</entry></row><row><entry></entry></row><row><entry>YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKT</entry></row><row><entry></entry></row><row><entry>SEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI</entry></row><row><entry></entry></row><row><entry>NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT</entry></row><row><entry></entry></row><row><entry>TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT</entry></row><row><entry></entry></row><row><entry>DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKY</entry></row><row><entry></entry></row><row><entry>LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLA</entry></row><row><entry></entry></row><row><entry>QISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSED</entry></row><row><entry></entry></row><row><entry>KANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF</entry></row><row><entry></entry></row><row><entry>ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENK</entry></row><row><entry></entry></row><row><entry>GEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN</entry></row><row><entry></entry></row><row><entry>GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSI</entry></row><row><entry></entry></row><row><entry>DEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR</entry></row><row><entry></entry></row><row><entry>PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIA</entry></row><row><entry></entry></row><row><entry>NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEI</entry></row><row><entry></entry></row><row><entry>NLLLKEKANDVHILSI<u style="single"><b>A</b></u>RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMK</entry></row><row><entry></entry></row><row><entry>TNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYN</entry></row><row><entry></entry></row><row><entry>AIVVF<u style="single"><b>E</b></u>DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG</entry></row><row><entry></entry></row><row><entry>VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYE</entry></row><row><entry></entry></row><row><entry>SVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR</entry></row><row><entry></entry></row><row><entry>LINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD</entry></row><row><entry></entry></row><row><entry>KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNM</entry></row><row><entry></entry></row><row><entry>PQDA<u style="single"><b>D</b></u>ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN</entry></row></tbody></tgroup></table></tables><br /><i>Francisella novicida </i>Cpf1 E1006A (SEQ ID NO: 11) (D917, A1006, and D1255 are bolded and underlined)

0257<tables id="TABLE-US-00009" num="00009"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 11)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKA</entry></row><row><entry></entry></row><row><entry>KQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKS</entry></row><row><entry></entry></row><row><entry>AKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGI</entry></row><row><entry></entry></row><row><entry>ELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSII</entry></row><row><entry></entry></row><row><entry>YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKT</entry></row><row><entry></entry></row><row><entry>SEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI</entry></row><row><entry></entry></row><row><entry>NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT</entry></row><row><entry></entry></row><row><entry>TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT</entry></row><row><entry></entry></row><row><entry>DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKY</entry></row><row><entry></entry></row><row><entry>LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLA</entry></row><row><entry></entry></row><row><entry>QISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSED</entry></row><row><entry></entry></row><row><entry>KANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF</entry></row><row><entry></entry></row><row><entry>ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENK</entry></row><row><entry></entry></row><row><entry>GEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN</entry></row><row><entry></entry></row><row><entry>GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSI</entry></row><row><entry></entry></row><row><entry>DEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR</entry></row><row><entry></entry></row><row><entry>PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIA</entry></row><row><entry></entry></row><row><entry>NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEI</entry></row><row><entry></entry></row><row><entry>NLLLKEKANDVHILSI<u style="single"><b>D</b></u>RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMK</entry></row><row><entry></entry></row><row><entry>TNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYN</entry></row><row><entry></entry></row><row><entry>AIVVF<u style="single"><b>A</b></u>DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG</entry></row><row><entry></entry></row><row><entry>VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYE</entry></row><row><entry></entry></row><row><entry>SVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR</entry></row><row><entry></entry></row><row><entry>LINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD</entry></row><row><entry></entry></row><row><entry>KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNM</entry></row><row><entry></entry></row><row><entry>PQDA<u style="single"><b>D</b></u>ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN</entry></row></tbody></tgroup></table></tables><br /><i>Francisella novicida </i>Cpf1 D1255A (SEQ ID NO: 12) (D917, E1006, and A1255 are bolded and underlined)

0258<tables id="TABLE-US-00010" num="00010"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 12)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKA</entry></row><row><entry></entry></row><row><entry>KQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKS</entry></row><row><entry></entry></row><row><entry>AKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGI</entry></row><row><entry></entry></row><row><entry>ELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSII</entry></row><row><entry></entry></row><row><entry>YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKT</entry></row><row><entry></entry></row><row><entry>SEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI</entry></row><row><entry></entry></row><row><entry>NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT</entry></row><row><entry></entry></row><row><entry>TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT</entry></row><row><entry></entry></row><row><entry>DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKY</entry></row><row><entry></entry></row><row><entry>LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLA</entry></row><row><entry></entry></row><row><entry>QISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSED</entry></row><row><entry></entry></row><row><entry>KANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF</entry></row><row><entry></entry></row><row><entry>ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENK</entry></row><row><entry></entry></row><row><entry>GEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN</entry></row><row><entry></entry></row><row><entry>GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSI</entry></row><row><entry></entry></row><row><entry>DEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR</entry></row><row><entry></entry></row><row><entry>PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIA</entry></row><row><entry></entry></row><row><entry>NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEI</entry></row><row><entry></entry></row><row><entry>NLLLKEKANDVHILSI<u style="single"><b>D</b></u>RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMK</entry></row><row><entry></entry></row><row><entry>TNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYN</entry></row><row><entry></entry></row><row><entry>AIVVF<u style="single"><b>E</b></u>DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG</entry></row><row><entry></entry></row><row><entry>VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYE</entry></row><row><entry></entry></row><row><entry>SVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR</entry></row><row><entry></entry></row><row><entry>LINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD</entry></row><row><entry></entry></row><row><entry>KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNM</entry></row><row><entry></entry></row><row><entry>PQDA<u style="single"><b>A</b></u>ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN</entry></row></tbody></tgroup></table></tables><br /><i>Francisella novicida </i>Cpf1 D917A/E1006A (SEQ ID NO: 13) (A917, A1006, and D1255 are bolded and underlined)

0259<tables id="TABLE-US-00011" num="00011"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 13)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKA</entry></row><row><entry></entry></row><row><entry>KQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKS</entry></row><row><entry></entry></row><row><entry>AKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGI</entry></row><row><entry></entry></row><row><entry>ELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSII</entry></row><row><entry></entry></row><row><entry>YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKT</entry></row><row><entry></entry></row><row><entry>SEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI</entry></row><row><entry></entry></row><row><entry>NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT</entry></row><row><entry></entry></row><row><entry>TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT</entry></row><row><entry></entry></row><row><entry>DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKY</entry></row><row><entry></entry></row><row><entry>LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLA</entry></row><row><entry></entry></row><row><entry>QISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSED</entry></row><row><entry></entry></row><row><entry>KANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF</entry></row><row><entry></entry></row><row><entry>ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENK</entry></row><row><entry></entry></row><row><entry>GEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN</entry></row><row><entry></entry></row><row><entry>GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSI</entry></row><row><entry></entry></row><row><entry>DEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR</entry></row><row><entry></entry></row><row><entry>PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIA</entry></row><row><entry></entry></row><row><entry>NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEI</entry></row><row><entry></entry></row><row><entry>NLLLKEKANDVHILSI<u style="single"><b>A</b></u>RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMK</entry></row><row><entry></entry></row><row><entry>TNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYN</entry></row><row><entry></entry></row><row><entry>AIVVF<u style="single"><b>A</b></u>DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG</entry></row><row><entry></entry></row><row><entry>VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYE</entry></row><row><entry></entry></row><row><entry>SVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR</entry></row><row><entry></entry></row><row><entry>LINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD</entry></row><row><entry></entry></row><row><entry>KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNM</entry></row><row><entry></entry></row><row><entry>PQDA<u style="single"><b>D</b></u>ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN</entry></row></tbody></tgroup></table></tables><br /><i>Francisella novicida </i>Cpf1 D917A/D1255A (SEQ ID NO: 14) (A917, E1006, and A1255 are bolded and underlined)

0260<tables id="TABLE-US-00012" num="00012"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 14)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKA</entry></row><row><entry></entry></row><row><entry>KQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKS</entry></row><row><entry></entry></row><row><entry>AKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGI</entry></row><row><entry></entry></row><row><entry>ELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSII</entry></row><row><entry></entry></row><row><entry>YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKT</entry></row><row><entry></entry></row><row><entry>SEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI</entry></row><row><entry></entry></row><row><entry>NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT</entry></row><row><entry></entry></row><row><entry>TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT</entry></row><row><entry></entry></row><row><entry>DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKY</entry></row><row><entry></entry></row><row><entry>LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLA</entry></row><row><entry></entry></row><row><entry>QISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSED</entry></row><row><entry></entry></row><row><entry>KANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF</entry></row><row><entry></entry></row><row><entry>ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENK</entry></row><row><entry></entry></row><row><entry>GEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN</entry></row><row><entry></entry></row><row><entry>GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSI</entry></row><row><entry></entry></row><row><entry>DEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR</entry></row><row><entry></entry></row><row><entry>PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIA</entry></row><row><entry></entry></row><row><entry>NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEI</entry></row><row><entry></entry></row><row><entry>NLLLKEKANDVHILSI<u style="single"><b>A</b></u>RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMK</entry></row><row><entry></entry></row><row><entry>TNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYN</entry></row><row><entry></entry></row><row><entry>AIVVF<u style="single"><b>E</b></u>DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG</entry></row><row><entry></entry></row><row><entry>VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYE</entry></row><row><entry></entry></row><row><entry>SVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR</entry></row><row><entry></entry></row><row><entry>LINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD</entry></row><row><entry></entry></row><row><entry>KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNM</entry></row><row><entry></entry></row><row><entry>PQDA<u style="single"><b>A</b></u>ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN</entry></row></tbody></tgroup></table></tables><br /><i>Francisella novicida </i>Cpf1 E1006A/D1255A (SEQ ID NO: 15) (D917, A1006, and A1255 are bolded and underlined)

0261<tables id="TABLE-US-00013" num="00013"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 15)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKA</entry></row><row><entry></entry></row><row><entry>KQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKS</entry></row><row><entry></entry></row><row><entry>AKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGI</entry></row><row><entry></entry></row><row><entry>ELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSII</entry></row><row><entry></entry></row><row><entry>YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKT</entry></row><row><entry></entry></row><row><entry>SEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI</entry></row><row><entry></entry></row><row><entry>NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT</entry></row><row><entry></entry></row><row><entry>TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT</entry></row><row><entry></entry></row><row><entry>DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKY</entry></row><row><entry></entry></row><row><entry>LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLA</entry></row><row><entry></entry></row><row><entry>QISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSED</entry></row><row><entry></entry></row><row><entry>KANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF</entry></row><row><entry></entry></row><row><entry>ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENK</entry></row><row><entry></entry></row><row><entry>GEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN</entry></row><row><entry></entry></row><row><entry>GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSI</entry></row><row><entry></entry></row><row><entry>DEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR</entry></row><row><entry></entry></row><row><entry>PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIA</entry></row><row><entry></entry></row><row><entry>NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEI</entry></row><row><entry></entry></row><row><entry>NLLLKEKANDVHILSI<u style="single"><b>D</b></u>RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMK</entry></row><row><entry></entry></row><row><entry>TNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYN</entry></row><row><entry></entry></row><row><entry>AIVVF<u style="single"><b>A</b></u>DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG</entry></row><row><entry></entry></row><row><entry>VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYE</entry></row><row><entry></entry></row><row><entry>SVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR</entry></row><row><entry></entry></row><row><entry>LINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD</entry></row><row><entry></entry></row><row><entry>KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNM</entry></row><row><entry></entry></row><row><entry>PQDA<u style="single"><b>A</b></u>ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN</entry></row></tbody></tgroup></table></tables><br /><i>Francisella novicida </i>Cpf1 D917A/E1006A/D1255A (SEQ ID NO: 16) (A917, A1006, and A1255 are bolded and underlined)

0262<tables id="TABLE-US-00014" num="00014"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 16)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKA</entry></row><row><entry></entry></row><row><entry>KQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKS</entry></row><row><entry></entry></row><row><entry>AKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGI</entry></row><row><entry></entry></row><row><entry>ELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSII</entry></row><row><entry></entry></row><row><entry>YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKT</entry></row><row><entry></entry></row><row><entry>SEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI</entry></row><row><entry></entry></row><row><entry>NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT</entry></row><row><entry></entry></row><row><entry>TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT</entry></row><row><entry></entry></row><row><entry>DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKY</entry></row><row><entry></entry></row><row><entry>LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLA</entry></row><row><entry></entry></row><row><entry>QISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSED</entry></row><row><entry></entry></row><row><entry>KANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF</entry></row><row><entry></entry></row><row><entry>ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENK</entry></row><row><entry></entry></row><row><entry>GEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN</entry></row><row><entry></entry></row><row><entry>GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSI</entry></row><row><entry></entry></row><row><entry>DEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR</entry></row><row><entry></entry></row><row><entry>PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIA</entry></row><row><entry></entry></row><row><entry>NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEI</entry></row><row><entry></entry></row><row><entry>NLLLKEKANDVHILSI<u style="single"><b>A</b></u>RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMK</entry></row><row><entry></entry></row><row><entry>TNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYN</entry></row><row><entry></entry></row><row><entry>AIVVF<u style="single"><b>A</b></u>DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG</entry></row><row><entry></entry></row><row><entry>VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYE</entry></row><row><entry></entry></row><row><entry>SVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR</entry></row><row><entry></entry></row><row><entry>LINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD</entry></row><row><entry></entry></row><row><entry>KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNM</entry></row><row><entry></entry></row><row><entry>PQDA<u style="single"><b>A</b></u>ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN</entry></row></tbody></tgroup></table></tables>

0263In some embodiments, the nucleic acid programmable DNA binding protein is a Cpf1 protein from an <i>Acidaminococcus </i>species (AsCpf1). Cpf1 proteins form <i>Acidaminococcus </i>species have been described previously and would be apparent to the skilled artisan. Exemplary <i>Acidaminococcus </i>Cpf1 proteins (AsCpf1) include, without limitation, any of the AsCpf1 proteins provided herein.

0264Wild-type AsCpf1—Residue R912 is indicated in bold underlining and residues 661-667 are indicated in italics and underlining.

0265<tables id="TABLE-US-00015" num="00015"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 17)</entry></row><row><entry>TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELK</entry></row><row><entry></entry></row><row><entry>PIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQAT</entry></row><row><entry></entry></row><row><entry>YRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTT</entry></row><row><entry></entry></row><row><entry>TEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKF</entry></row><row><entry></entry></row><row><entry>KENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLT</entry></row><row><entry></entry></row><row><entry>QTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHR</entry></row><row><entry></entry></row><row><entry>FIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEA</entry></row><row><entry></entry></row><row><entry>LFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKI</entry></row><row><entry></entry></row><row><entry>TKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALD</entry></row><row><entry></entry></row><row><entry>QPLPTTMLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARL</entry></row><row><entry></entry></row><row><entry>TGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEK</entry></row><row><entry></entry></row><row><entry>NNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPD</entry></row><row><entry></entry></row><row><entry>AAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEK</entry></row><row><entry></entry></row><row><entry>EPKKFQTAYA<u style="single"><i>KKTGDQK</i></u>GYREALCKWIDFTRDFLSKYTKTTSIDLSSLRP</entry></row><row><entry></entry></row><row><entry>SSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDF</entry></row><row><entry></entry></row><row><entry>AKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAH</entry></row><row><entry></entry></row><row><entry>RLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVI</entry></row><row><entry></entry></row><row><entry>TKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHP</entry></row><row><entry></entry></row><row><entry>ETPIIGIDRGE<u style="single"><b>R</b></u>NLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKE</entry></row><row><entry></entry></row><row><entry>RVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFK</entry></row><row><entry></entry></row><row><entry>SKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFT</entry></row><row><entry></entry></row><row><entry>SFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEG</entry></row><row><entry></entry></row><row><entry>FDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAK</entry></row><row><entry></entry></row><row><entry>GTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNIL</entry></row><row><entry></entry></row><row><entry>PKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFD</entry></row><row><entry></entry></row><row><entry>SRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLA</entry></row><row><entry></entry></row><row><entry>YIQELRN</entry></row></tbody></tgroup></table></tables>

0266AsCpf1(R912A)—Residue A912 is indicated in bold underlining and residues 661-667 are indicated in italics and underlining.

0267<tables id="TABLE-US-00016" num="00016"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 19)</entry></row><row><entry>TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELK</entry></row><row><entry></entry></row><row><entry>PIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQAT</entry></row><row><entry></entry></row><row><entry>YRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTT</entry></row><row><entry></entry></row><row><entry>TEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKF</entry></row><row><entry></entry></row><row><entry>KENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLT</entry></row><row><entry></entry></row><row><entry>QTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHR</entry></row><row><entry></entry></row><row><entry>FIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEA</entry></row><row><entry></entry></row><row><entry>LFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKI</entry></row><row><entry></entry></row><row><entry>TKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALD</entry></row><row><entry></entry></row><row><entry>QPLPTTMLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARL</entry></row><row><entry></entry></row><row><entry>TGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEK</entry></row><row><entry></entry></row><row><entry>NNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPD</entry></row><row><entry></entry></row><row><entry>AAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEK</entry></row><row><entry></entry></row><row><entry>EPKKFQTAYA<u style="single"><i>KKTGDQK</i></u>GYREALCKWIDFTRDFLSKYTKTTSIDLSSLRP</entry></row><row><entry></entry></row><row><entry>SSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDF</entry></row><row><entry></entry></row><row><entry>AKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAH</entry></row><row><entry></entry></row><row><entry>RLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVI</entry></row><row><entry></entry></row><row><entry>TKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHP</entry></row><row><entry></entry></row><row><entry>ETPIIGIDRGE<u style="single"><b>A</b></u>NLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKE</entry></row><row><entry></entry></row><row><entry>RVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFK</entry></row><row><entry></entry></row><row><entry>SKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFT</entry></row><row><entry></entry></row><row><entry>SFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEG</entry></row><row><entry></entry></row><row><entry>FDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAK</entry></row><row><entry></entry></row><row><entry>GTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNIL</entry></row><row><entry></entry></row><row><entry>PKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFD</entry></row><row><entry></entry></row><row><entry>SRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLA</entry></row><row><entry></entry></row><row><entry>YIQELRN</entry></row></tbody></tgroup></table></tables>

0268In some embodiments, the nucleic acid programmable DNA binding protein is a Cpf1 protein from a Lachnospiraceae species (LbCpf1). Cpf1 proteins form Lachnospiraceae species have been described previously have been described previously and would be apparent to the skilled artisan. Exemplary Lachnospiraceae Cpf1 proteins (LbCpf1) include, without limitation, any of the LbCpf1 proteins provided herein.

0269In some embodiments, the LbCpf1 is a nickase. In some embodiments, the LbCpf1 nickase comprises an R836X mutant relative to SEQ ID NO: 18, wherein X is any amino acid except for R. In some embodiments, the LbCpf1 nickase comprises R836A mutant relative to SEQ ID NO: 18. In some embodiments, the LbCpf1 is a nuclease inactive LbCpf1 (dLbCpf1). In some embodiments, the dLbCpf1 comprises a D832X mutant relative to SEQ ID NO: 18, wherein X is any amino acid except for D. In some embodiments, the dLbCpf1 comprises a D832A mutant relative to SEQ ID NO: 18. Additional dCpf1 proteins have been described in the art, for example, in Li et al. “Base editing with a Cpf1-cytidine deaminase fusion” <i>Nature Biotechnology</i>; March 2018 DOI: 10.1038/nbt.4102; the entire contents of which are incorporated herein by reference. In some embodiments, the dCpf1 comprises 1, 2, or 3 of the point mutations D832A, E1006A, D1125A of the Cpf1 described in Li et al.

0270Wild-type LbCpf1—Residues R836 and R1138 is indicated in bold underlining.

0271<tables id="TABLE-US-00017" num="00017"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 18)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGV</entry></row><row><entry></entry></row><row><entry>KKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEIN</entry></row><row><entry></entry></row><row><entry>LRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTA</entry></row><row><entry></entry></row><row><entry>FTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKH</entry></row><row><entry></entry></row><row><entry>EVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGE</entry></row><row><entry></entry></row><row><entry>KIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEV</entry></row><row><entry></entry></row><row><entry>LEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKD</entry></row><row><entry></entry></row><row><entry>IFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQL</entry></row><row><entry></entry></row><row><entry>QEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKND</entry></row><row><entry></entry></row><row><entry>AVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKV</entry></row><row><entry></entry></row><row><entry>DHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYG</entry></row><row><entry></entry></row><row><entry>SKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSK</entry></row><row><entry></entry></row><row><entry>KWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWS</entry></row><row><entry></entry></row><row><entry>NAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLY</entry></row><row><entry></entry></row><row><entry>MFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRAS</entry></row><row><entry></entry></row><row><entry>LKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPI</entry></row><row><entry></entry></row><row><entry>AINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGE<u style="single"><b>R</b></u>NLLYIVVVDGKGNI</entry></row><row><entry></entry></row><row><entry>VEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELK</entry></row><row><entry></entry></row><row><entry>AGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKML</entry></row><row><entry></entry></row><row><entry>IDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWL</entry></row><row><entry></entry></row><row><entry>TSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYK</entry></row><row><entry></entry></row><row><entry>NFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFN</entry></row><row><entry></entry></row><row><entry>KYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQM<u style="single"><b>R</b></u>NSITGRTDVDFL</entry></row><row><entry></entry></row><row><entry>ISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKK</entry></row><row><entry></entry></row><row><entry>AEDEKLDKVKIAISNKEWLEYAQTSVKH</entry></row></tbody></tgroup></table></tables>

0272LbCpf1 (R836A)—Residue A836 is indicated in bold underlining.

0273<tables id="TABLE-US-00018" num="00018"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 20)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGV</entry></row><row><entry></entry></row><row><entry>KKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEIN</entry></row><row><entry></entry></row><row><entry>LRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTA</entry></row><row><entry></entry></row><row><entry>FTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKH</entry></row><row><entry></entry></row><row><entry>EVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGE</entry></row><row><entry></entry></row><row><entry>KIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEV</entry></row><row><entry></entry></row><row><entry>LEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKD</entry></row><row><entry></entry></row><row><entry>IFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQL</entry></row><row><entry></entry></row><row><entry>QEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKND</entry></row><row><entry></entry></row><row><entry>AVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKV</entry></row><row><entry></entry></row><row><entry>DHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYG</entry></row><row><entry></entry></row><row><entry>SKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSK</entry></row><row><entry></entry></row><row><entry>KWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWS</entry></row><row><entry></entry></row><row><entry>NAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLY</entry></row><row><entry></entry></row><row><entry>MFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRAS</entry></row><row><entry></entry></row><row><entry>LKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPI</entry></row><row><entry></entry></row><row><entry>AINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGE<u style="single"><b>A</b></u>NLLYIVVVDGKGNI</entry></row><row><entry></entry></row><row><entry>VEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELK</entry></row><row><entry></entry></row><row><entry>AGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKML</entry></row><row><entry></entry></row><row><entry>IDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWL</entry></row><row><entry></entry></row><row><entry>TSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYK</entry></row><row><entry></entry></row><row><entry>NFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFN</entry></row><row><entry></entry></row><row><entry>KYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFL</entry></row><row><entry></entry></row><row><entry>ISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKK</entry></row><row><entry></entry></row><row><entry>AEDEKLDKVKIAISNKEWLEYAQTSVKH</entry></row></tbody></tgroup></table></tables>

0274LbCpf1 (R1138A)—Residue A1138 is indicated in bold underlining.

0275<tables id="TABLE-US-00019" num="00019"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 21)</entry></row><row><entry>MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGV</entry></row><row><entry></entry></row><row><entry>KKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEIN</entry></row><row><entry></entry></row><row><entry>LRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTA</entry></row><row><entry></entry></row><row><entry>FTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKH</entry></row><row><entry></entry></row><row><entry>EVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGE</entry></row><row><entry></entry></row><row><entry>KIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEV</entry></row><row><entry></entry></row><row><entry>LEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKD</entry></row><row><entry></entry></row><row><entry>IFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQL</entry></row><row><entry></entry></row><row><entry>QEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKND</entry></row><row><entry></entry></row><row><entry>AVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKV</entry></row><row><entry></entry></row><row><entry>DHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYG</entry></row><row><entry></entry></row><row><entry>SKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSK</entry></row><row><entry></entry></row><row><entry>KWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWS</entry></row><row><entry></entry></row><row><entry>NAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLY</entry></row><row><entry></entry></row><row><entry>MFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRAS</entry></row><row><entry></entry></row><row><entry>LKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPI</entry></row><row><entry></entry></row><row><entry>AINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNI</entry></row><row><entry></entry></row><row><entry>VEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELK</entry></row><row><entry></entry></row><row><entry>AGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKML</entry></row><row><entry></entry></row><row><entry>IDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWL</entry></row><row><entry></entry></row><row><entry>TSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYK</entry></row><row><entry></entry></row><row><entry>NFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFN</entry></row><row><entry></entry></row><row><entry>KYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQM<u style="single"><b>A</b></u>NSITGRTDVDFL</entry></row><row><entry></entry></row><row><entry>ISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKK</entry></row><row><entry></entry></row><row><entry>AEDEKLDKVKIAISNKEWLEYAQTSVKH</entry></row></tbody></tgroup></table></tables>

0276In some embodiments, the Cpf1 protein is a crippled Cpf1 protein. As used herein a “crippled Cpf1” protein is a Cpf1 protein having diminished nuclease activity as compared to a wild-type Cpf1 protein. In some embodiments, the crippled Cpf1 protein preferentially cuts the target strand more efficiently than the non-target strand. For example, the Cpf1 protein preferentially cuts the strand of a duplexed nucleic acid molecule in which a nucleotide to be edited resides. In some embodiments, the crippled Cpf1 protein preferentially cuts the non-target strand more efficiently than the target strand. For example, the Cpf1 protein preferentially cuts the strand of a duplexed nucleic acid molecule in which a nucleotide to be edited does not reside. In some embodiments, the crippled Cpf1 protein preferentially cuts the target strand at least 5% more efficiently than it cuts the non-target strand. In some embodiments, the crippled Cpf1 protein preferentially cuts the target strand at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 100% more efficiently than it cuts the non-target strand.

0277In some embodiments, a crippled Cpf1 protein is a non-naturally occurring Cpf1 protein. In some embodiments, the crippled Cpf1 protein comprises one or more mutations relative to a wild-type Cpf1 protein. In some embodiments, the crippled Cpf1 protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations relative to a wild-type Cpf1 protein. In some embodiments, the crippled Cpf1 protein comprises an R836A mutation as set forth in SEQ ID NO: 18, or in a corresponding amino acid in another Cpf1 protein. It should be appreciated that a Cpf1 comprising a homologous residue (e.g., a corresponding amino acid) to R836A of SEQ ID NO: 18 could also be mutated to achieve similar results. In some embodiments, the crippled Cpf1 protein comprises a R1138A mutation as set forth in SEQ ID NO: 18, or in a corresponding amino acid in another Cpf1 protein. In some embodiments, the crippled Cpf1 protein comprises an R912A mutation mutation as set forth in SEQ ID NO: 17, or in a corresponding amino acid in another Cpf1 protein. Without wishing to be bound by any particular theory, residue R836 of SEQ ID NO: 18 (LbCpf1) and residue R912 of SEQ ID NO: 17 (AsCpf1) are examples of corresponding (e.g., homologous) residues. For example, a portion of the alignment between SEQ ID NO: 17 and 18 shows that R912 and R836 are corresponding residues.

0278<tables id="TABLE-US-00020" num="00020"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="28pt" align="left" /><colspec colname="2" colwidth="259pt" align="left" /><colspec colname="3" colwidth="154pt" align="left" /><tbody valign="top"><row><entry>AsCpf1</entry><entry>YQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQ--</entry><entry /></row><row><entry>LbCpf1</entry><entry>KCPKN-IFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINN</entry></row><row><entry /><entry>    *   *:* .*.. **.. :  :**********:**.*:*..*:*:** *** *</entry></row></tbody></tgroup></table></tables>

0279In some embodiments, any of the Cpf1 proteins provided herein comprises one or more amino acid deletions. In some embodiments, any of the Cpf1 proteins provided herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid deletions. Without wishing to be bound by any particular theory, there is a helical region in Cpf1, which includes residues 661-667 of AsCpf1 (SEQ ID NO: 17), that may obstruct the function of a deaminase (e.g., APOBEC) that is fused to the Cpf1. This region comprises the amino acid sequence KKTGDQK. Accordingly, aspects of the disclosure provide Cpf1 proteins comprising mutations (e.g., deletions) that disrupt this helical region in Cpf1. In some embodiments, the Cpf1 protein comprises one or more deletions of the following residues in SEQ ID NO: 17, or one or more corresponding deletions in another Cpf1 protein: K661, K662, T663, G664, D665, Q666, and K667. In some embodiments, the Cpf1 protein comprises a T663 and a D665 deletion in SEQ ID NO: 17, or corresponding deletions in another Cpf1 protein. In some embodiments, the Cpf1 protein comprises a K662, T663, D665, and Q666 deletion in SEQ ID NO: 17, or corresponding deletions in another Cpf1 protein. In some embodiments, the Cpf1 protein comprises a K661, K662, T663, D665, Q666 and K667 deletion in SEQ ID NO: 17, or corresponding deletions in another Cpf1 protein.

0280AsCpf1 (Deleted T663 and D665)

0281<tables id="TABLE-US-00021" num="00021"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>AsCpf1 (deleted T663 and D665)</entry></row><row><entry>(SEQ ID NO: 22)</entry></row><row><entry>TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELK</entry></row><row><entry></entry></row><row><entry>PIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQAT</entry></row><row><entry></entry></row><row><entry>YRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTT</entry></row><row><entry></entry></row><row><entry>TEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKF</entry></row><row><entry></entry></row><row><entry>KENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLT</entry></row><row><entry></entry></row><row><entry>QTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHR</entry></row><row><entry></entry></row><row><entry>FIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEA</entry></row><row><entry></entry></row><row><entry>LFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKI</entry></row><row><entry></entry></row><row><entry>TKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALD</entry></row><row><entry></entry></row><row><entry>QPLPTTMLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARL</entry></row><row><entry></entry></row><row><entry>TGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEK</entry></row><row><entry></entry></row><row><entry>NNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPD</entry></row><row><entry></entry></row><row><entry>AAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEK</entry></row><row><entry></entry></row><row><entry>EPKKFQTAYAKKGQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSS</entry></row><row><entry></entry></row><row><entry>QYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAK</entry></row><row><entry></entry></row><row><entry>GHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRL</entry></row><row><entry></entry></row><row><entry>GEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITK</entry></row><row><entry></entry></row><row><entry>EVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPET</entry></row><row><entry></entry></row><row><entry>PIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERV</entry></row><row><entry></entry></row><row><entry>AARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSK</entry></row><row><entry></entry></row><row><entry>RTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSF</entry></row><row><entry></entry></row><row><entry>AKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFD</entry></row><row><entry></entry></row><row><entry>FLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGT</entry></row><row><entry></entry></row><row><entry>PFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPK</entry></row><row><entry></entry></row><row><entry>LLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSR</entry></row><row><entry></entry></row><row><entry>FQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI</entry></row><row><entry></entry></row><row><entry>QELRN</entry></row></tbody></tgroup></table></tables>

0282AsCpf1 (Deleted K662, T663, D665, and Q666)

0283<tables id="TABLE-US-00022" num="00022"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>AsCpf1 (deleted K662, T663, D665, and Q666)</entry></row><row><entry>(SEQ ID NO: 23)</entry></row><row><entry>TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELK</entry></row><row><entry></entry></row><row><entry>PIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQAT</entry></row><row><entry></entry></row><row><entry>YRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTT</entry></row><row><entry></entry></row><row><entry>TEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKF</entry></row><row><entry></entry></row><row><entry>KENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLT</entry></row><row><entry></entry></row><row><entry>QTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHR</entry></row><row><entry></entry></row><row><entry>FIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEA</entry></row><row><entry></entry></row><row><entry>LFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKI</entry></row><row><entry></entry></row><row><entry>TKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALD</entry></row><row><entry></entry></row><row><entry>QPLPTTMLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARL</entry></row><row><entry></entry></row><row><entry>TGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEK</entry></row><row><entry></entry></row><row><entry>NNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPD</entry></row><row><entry></entry></row><row><entry>AAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEK</entry></row><row><entry></entry></row><row><entry>EPKKFQTAYAKGKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQY</entry></row><row><entry></entry></row><row><entry>KDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGH</entry></row><row><entry></entry></row><row><entry>HGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGE</entry></row><row><entry></entry></row><row><entry>KMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEV</entry></row><row><entry></entry></row><row><entry>SHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPI</entry></row><row><entry></entry></row><row><entry>IGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAA</entry></row><row><entry></entry></row><row><entry>RQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRT</entry></row><row><entry></entry></row><row><entry>GIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK</entry></row><row><entry></entry></row><row><entry>MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFL</entry></row><row><entry></entry></row><row><entry>HYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPF</entry></row><row><entry></entry></row><row><entry>IAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLL</entry></row><row><entry></entry></row><row><entry>ENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQ</entry></row><row><entry></entry></row><row><entry>NPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQE</entry></row><row><entry></entry></row><row><entry>LRN</entry></row></tbody></tgroup></table></tables>

0284AsCpf1 (Deleted K661, K662, T663, D665, Q666, and K667)

0285<tables id="TABLE-US-00023" num="00023"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>AsCpf1 (deleted K661, K662, T663, D665, Q666, and</entry></row><row><entry>K667)</entry></row><row><entry>(SEQ ID NO: 24)</entry></row><row><entry>TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELK</entry></row><row><entry></entry></row><row><entry>PIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQAT</entry></row><row><entry></entry></row><row><entry>YRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTT</entry></row><row><entry></entry></row><row><entry>TEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKF</entry></row><row><entry></entry></row><row><entry>KENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLT</entry></row><row><entry></entry></row><row><entry>QTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHR</entry></row><row><entry></entry></row><row><entry>FIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEA</entry></row><row><entry></entry></row><row><entry>LFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKI</entry></row><row><entry></entry></row><row><entry>TKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALD</entry></row><row><entry></entry></row><row><entry>QPLPTTMLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARL</entry></row><row><entry></entry></row><row><entry>TGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEK</entry></row><row><entry></entry></row><row><entry>NNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPD</entry></row><row><entry></entry></row><row><entry>AAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEK</entry></row><row><entry></entry></row><row><entry>EPKKFQTAYAGGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKD</entry></row><row><entry></entry></row><row><entry>LGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHG</entry></row><row><entry></entry></row><row><entry>KPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM</entry></row><row><entry></entry></row><row><entry>LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSH</entry></row><row><entry></entry></row><row><entry>EIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIG</entry></row><row><entry></entry></row><row><entry>IDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQ</entry></row><row><entry></entry></row><row><entry>AWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGI</entry></row><row><entry></entry></row><row><entry>AEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMG</entry></row><row><entry></entry></row><row><entry>TQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHY</entry></row><row><entry></entry></row><row><entry>DVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIA</entry></row><row><entry></entry></row><row><entry>GKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLEN</entry></row><row><entry></entry></row><row><entry>DDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNP</entry></row><row><entry></entry></row><row><entry>EWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELR</entry></row><row><entry></entry></row><row><entry>N</entry></row></tbody></tgroup></table></tables>

0286In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a nucleic acid programmable DNA binding protein that does not require a canonical (NGG) PAM sequence in the target sequence. In some embodiments, the napDNAbp is an Argonaute protein. One example of such a nucleic acid programmable DNA binding protein is an Argonaute protein from <i>Natronobacterium gregoryi </i>(NgAgo). NgAgo is a ssDNA-guided endonuclease. NgAgo binds 5′-phosphorylated ssDNA of ˜24 nucleotides (gDNA) in length to guide it to a target site and makes DNA double-strand breaks at the gDNA site. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM). Using a nuclease inactive NgAgo (dNgAgo) can greatly expand the bases that may be targeted. The characterization and use of NgAgo have been described in Gao et al., <i>Nat. Biotechnol., </i>2016 July; 34(7):768-73. PubMed PMID: 27136078; Swarts et al., <i>Nature </i>507(7491) (2014):258-61; and Swarts et al., <i>Nucleic Acids Res. </i>43(10) (2015):5120-9, each of which is incorporated herein by reference. The sequence of <i>Natronobacterium gregoryi </i>Argonaute is provided in SEQ ID NO: 25.

0287In some embodiments, the napDNAbp is an Argonaute protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring Argonaute protein. In some embodiments, the napDNAbp is a naturally-occurring Argonaute protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NO: 25. In some embodiments, the napDNAbp comprises an amino acid sequence of any one SEQ ID NO: 25.

0288<tables id="TABLE-US-00024" num="00024"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>Wild type <i>Natronobacterium</i> <i>gregoryi</i> Argonaute</entry></row><row><entry>(SEQ ID NO: 25)</entry></row><row><entry>MTVIDLDSTTTADELTSGHTYDISVTLTGVYDNTDEQHPRMSLAFEQDNG</entry></row><row><entry></entry></row><row><entry>ERRYITLWKNTTPKDVFTYDYATGSTYIFTNIDYEVKDGYENLTATYQTT</entry></row><row><entry></entry></row><row><entry>VENATAQEVGTTDEDETFAGGEPLDHHLDDALNETPDDAETESDSGHVMT</entry></row><row><entry></entry></row><row><entry>SFASRDQLPEWTLHTYTLTATDGAKTDTEYARRTLAYTVRQELYTDHDAA</entry></row><row><entry></entry></row><row><entry>PVATDGLMLLTPEPLGETPLDLDCGVRVEADETRTLDYTTAKDRLLAREL</entry></row><row><entry></entry></row><row><entry>VEEGLKRSLWDDYLVRGIDEVLSKEPVLTCDEFDLHERYDLSVEVGHSGR</entry></row><row><entry></entry></row><row><entry>AYLHINFRHRFVPKLTLADIDDDNIYPGLRVKTTYRPRRGHIVWGLRDEC</entry></row><row><entry></entry></row><row><entry>ATDSLNTLGNQSVVAYHRNNQTPINTDLLDAIEAADRRVVETRRQGHGDD</entry></row><row><entry></entry></row><row><entry>AVSFPQELLAVEPNTHQIKQFASDGFHQQARSKTRLSASRCSEKAQAFAE</entry></row><row><entry></entry></row><row><entry>RLDPVRLNGSTVEFSSEFFTGNNEQQLRLLYENGESVLTFRDGARGAHPD</entry></row><row><entry></entry></row><row><entry>ETFSKGIVNPPESFEVAVVLPEQQADTCKAQWDTMADLLNQAGAPPTRSE</entry></row><row><entry></entry></row><row><entry>TVQYDAFSSPESISLNVAGAIDPSEVDAAFVVLPPDQEGFADLASPTETY</entry></row><row><entry></entry></row><row><entry>DELKKALANMGIYSQMAYFDRFRDAKIFYTRNVALGLLAAAGGVAFTTEH</entry></row><row><entry></entry></row><row><entry>AMPGDADMFIGIDVSRSYPEDGASGQINIAATATAVYKDGTILGHSSTRP</entry></row><row><entry></entry></row><row><entry>QLGEKLQSTDVRDIMKNAILGYQQVTGESPTHIVIHRDGFMNEDLDPATE</entry></row><row><entry></entry></row><row><entry>FLNEQGVEYDIVEIRKQPQTRLLAVSDVQYDTPVKSIAAINQNEPRATVA</entry></row><row><entry></entry></row><row><entry>TFGAPEYLATRDGGGLPRPIQIERVAGETDIETLTRQVYLLSQSHIQVHN</entry></row><row><entry></entry></row><row><entry>STARLPITTAYADQASTHATKGYLVQTGAFESNVGFL</entry></row></tbody></tgroup></table></tables>

0289In some embodiments, the napDNAbp is a prokaryotic homolog of an Argonaute protein. Prokaryotic homologs of Argonaute proteins are known and have been described, for example, in Makarova K., et al., “Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements”, <i>Biol. Direct. </i>2009 Aug. 25; 4:29. doi: 10.1186/1745-6150-4-29, is incorporated herein by reference. In some embodiments, the napDNAbp is a <i>Marinitoga piezophila </i>Argunaute (MpAgo) protein. The CRISPR-associated <i>Marinitoga piezophila </i>Argonaute (MpAgo) protein cleaves single-stranded target sequences using 5′-phosphorylated guides. The 5′ guides are used by all known Argonautes. The crystal structure of an MpAgo-RNA complex shows a guide strand binding site comprising residues that block 5′ phosphate interactions. This data suggests the evolution of an Argonaute subclass with noncanonical specificity for a 5′-hydroxylated guide. See, e.g., Kaya et al., “A bacterial Argonaute with noncanonical guide RNA specificity”, <i>Proc Natl Acad Sci USA. </i>2016 Apr. 12; 113(15):4057-62, the entire contents of which are hereby incorporated by reference). It should be appreciated that other Argonaute proteins may be used in any of the fusion proteins (e.g., base editors) described herein, for example, to guide a deaminase (e.g., cytidine deaminase) to a target nucleic acid (e.g., ssRNA).

0290In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, without limitation, Cas9, Cpf1, C2c1, C2c2, and C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multisubunit effector complexes, while Class 2 systems have a single protein effector. Cas9 and Cpf1 are Class 2 effectors. In addition to Cas9 and Cpf1, three distinct Class 2 CRISPR-Cas systems (C2c1, C2c2, and C2c3) have been described by Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems”, <i>Mol. Cell, </i>2015 Nov. 5; 60(3): 385-397, the entire contents of which are herein incorporated by reference. Effectors of two of the systems, C2c1 and C2c3, contain RuvC-like endonuclease domains related to Cpf1. A third system, C2c2 contains an effector with two predicted HEPN RNase domains. Production of mature CRISPR RNA is tracrRNA-independent, unlike production of CRISPR RNA by C2c1. C2c1 depends on both CRISPR RNA and tracrRNA for DNA cleavage. Bacterial C2c2 has been shown to possess a unique RNase activity for CRISPR RNA maturation distinct from its RNA-activated single-stranded RNA degradation activity. These RNase functions are different from each other and from the CRISPR RNA-processing behavior of Cpf1. See, e.g., East-Seletsky, et al., “Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection”, <i>Nature, </i>2016 Oct. 13; 538(7624):270-273, the entire contents of which are hereby incorporated by reference. In vitro biochemical analysis of C2c2 in <i>Leptotrichia shahii </i>has shown that C2c2 is guided by a single CRISPR RNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. Catalytic residues in the two conserved HEPN domains mediate cleavage. Mutations in the catalytic residues generate catalytically inactive RNA-binding proteins. See e.g., Abudayyeh et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” <i>Science, </i>2016 Aug. 5; 353(6299), the entire contents of which are hereby incorporated by reference.

0291The crystal structure of <i>Alicyclobaccillus acidoterrastris </i>C2c1 (AacC2c1) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See, e.g., Liu et al., “C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism”, Mol. Cell, 2017 Jan. 19; 65(2):310-322, incorporated herein by reference. The crystal structure has also been reported for <i>Alicyclobacillus acidoterrestris </i>C2c1 bound to target DNAs as ternary complexes. See, e.g., Yang et al., “PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease”, <i>Cell, </i>2016 Dec. 15; 167(7):1814-1828, the entire contents of which are hereby incorporated by reference. Catalytically competent conformations of AacC2c1, both with target and non-target DNA strands, have been captured independently positioned within a single RuvC catalytic pocket, with C2c1-mediated cleavage resulting in a staggered seven-nucleotide break of target DNA. Structural comparisons between C2c1 ternary complexes and previously identified Cas9 and Cpf1 counterparts demonstrate the diversity of mechanisms used by CRISPR-Cas9 systems.

0292In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein is a C2c1, a C2c2, or a C2c3 protein. In some embodiments, the napDNAbp is a C2c1 protein. In some embodiments, the napDNAbp is a C2c2 protein. In some embodiments, the napDNAbp is a C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring C2c1, C2c2, or C2c3 protein. In some embodiments, the napDNAbp is a naturally-occurring C2c1, C2c2, or C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 26-28. In some embodiments, the napDNAbp comprises an amino acid sequence of any one SEQ ID NOs: 26-28. It should be appreciated that C2c1, C2c2, or C2c3 from other bacterial species may also be used in accordance with the present disclosure.

0000C2c1 (uniprot.org/uniprot/T0D7A2#)

0000sp|T0D7A2C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS=<i>Alicyclobacillus acidoterrestris </i>(strain ATCC 49025/DSM 3922/CIP 106132/NCIMB 13137/GD3B) GN=c2c1 PE=1 SV=1

0293<tables id="TABLE-US-00025" num="00025"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 26)</entry></row><row><entry>MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYR</entry></row><row><entry></entry></row><row><entry>RSPNGDGEQECDKTAEECKAELLERLRARQVENGHRGPAGSDDELLQLAR</entry></row><row><entry></entry></row><row><entry>QLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVR</entry></row><row><entry></entry></row><row><entry>MREAGEPGWEEEKEKAETRKSADRTADVLRALADFGLKPLMRVYTDSEMS</entry></row><row><entry></entry></row><row><entry>SVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQEYAKLVEQKN</entry></row><row><entry></entry></row><row><entry>RFEQKNFVGQEHLVHLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSD</entry></row><row><entry></entry></row><row><entry>KVFEKWGKLAPDAPFDLYDAEIKNVQRRNTRRFGSHDLFAKLAEPEYQAL</entry></row><row><entry></entry></row><row><entry>WREDASFLTRYAVYNSILRKLNHAKMFATFTLPDATAHPIWTRFDKLGGN</entry></row><row><entry></entry></row><row><entry>LHQYTFLFNEFGERRHAIRFHKLLKVENGVAREVDDVTVPISMSEQLDNL</entry></row><row><entry></entry></row><row><entry>LPRDPNEPIALYFRDYGAEQHFTGEFGGAKIQCRRDQLAHMHRRRGARDV</entry></row><row><entry></entry></row><row><entry>YLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHP</entry></row><row><entry></entry></row><row><entry>DDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPF</entry></row><row><entry></entry></row><row><entry>FFPIKGNDNLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLA</entry></row><row><entry></entry></row><row><entry>YLRLLVRCGSEDVGRRERSWAKLIEQPVDAANHMTPDWREAFENELQKLK</entry></row><row><entry></entry></row><row><entry>SLHGICSDKEWMDAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYAK</entry></row><row><entry></entry></row><row><entry>DVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREH</entry></row><row><entry></entry></row><row><entry>IDHAKEDRLKKLADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEEL</entry></row><row><entry></entry></row><row><entry>SEYQFNNDRPPSENNQLMQWSHRGVFQELINQAQVHDLLVGTMYAAFSSR</entry></row><row><entry></entry></row><row><entry>FDARTGAPGIRCRRVPARCTQEHNPEPFPWWLNKFVVEHTLDACPLRADD</entry></row><row><entry></entry></row><row><entry>LIPTGEGEIFVSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRLR</entry></row><row><entry></entry></row><row><entry>CDWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYERERGKKRRKV</entry></row><row><entry></entry></row><row><entry>FAQEKLSEEEAELLVEADEAREKSVVLMRDPSGIINRGNWTRQKEFWSMV</entry></row><row><entry></entry></row><row><entry>NQRIEGYLVKQIRSRVPLQDSACENTGDI</entry></row></tbody></tgroup></table></tables><br /> C2c2 (uniprot.org/uniprot/P0DOC6) <br /> >sp|P0DOC6|C2C2_LEPSD CRISPR-associated endoribonuclease C2c2 OS=<i>Leptotrichia shahii </i>(strain DSM 19757/CCUG 47503/CIP 107916/JCM 16776/LB37) GN=c2c2 PE=1 SV=1

0294<tables id="TABLE-US-00026" num="00026"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 27)</entry></row><row><entry>MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKEKID</entry></row><row><entry></entry></row><row><entry>NNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFL</entry></row><row><entry></entry></row><row><entry>ETEEVVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQE</entry></row><row><entry></entry></row><row><entry>NEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSL</entry></row><row><entry></entry></row><row><entry>YKIIEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIK</entry></row><row><entry></entry></row><row><entry>SNLEILGFVKFYLNVGGDKKKSKNKKMLVEKILNINVDLTVEDIADFVIK</entry></row><row><entry></entry></row><row><entry>ELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKHEKFKIERENK</entry></row><row><entry></entry></row><row><entry>KDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEI</entry></row><row><entry></entry></row><row><entry>FGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVR</entry></row><row><entry></entry></row><row><entry>LKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTV</entry></row><row><entry></entry></row><row><entry>NTDDFSRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDFFGGD</entry></row><row><entry></entry></row><row><entry>REKNYVLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRI</entry></row><row><entry></entry></row><row><entry>LHAISKERDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNI</entry></row><row><entry></entry></row><row><entry>ITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEK</entry></row><row><entry></entry></row><row><entry>IVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENI</entry></row><row><entry></entry></row><row><entry>IENYYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKM</entry></row><row><entry></entry></row><row><entry>NIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNA</entry></row><row><entry></entry></row><row><entry>VINKIRNRFFATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNL</entry></row><row><entry></entry></row><row><entry>EEFIQKMKEIEKDFDDFKIQTKKEIFNNYYEDIKNNILTEFKDDINGCDV</entry></row><row><entry></entry></row><row><entry>LEKKLEKIVIFDDETKFEIDKKSNILQDEQRKLSNINKKDLKKKVDQYIK</entry></row><row><entry></entry></row><row><entry>DKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYYPK</entry></row><row><entry></entry></row><row><entry>ERKNELYIYKKNLFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIR</entry></row><row><entry></entry></row><row><entry>KNKISEIDAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYK</entry></row><row><entry></entry></row><row><entry>SFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMH</entry></row><row><entry></entry></row><row><entry>YIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYK</entry></row><row><entry></entry></row><row><entry>KFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQI</entry></row><row><entry></entry></row><row><entry>DRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLIGNNDILE</entry></row><row><entry></entry></row><row><entry>RLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTL</entry></row></tbody></tgroup></table></tables>

0295C2c3, translated from >CEPX01008730.1 marine metagenome genome assembly TARA_037_MES_0.1-0.22, contig TARA_037_MES_0.1-0.22_scaffold22115_1, whole genome shotgun sequence.

0296<tables id="TABLE-US-00027" num="00027"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 28)</entry></row><row><entry>MRSNYHGGRNARQWRKQISGLARRTKETVFTYKFPLETDAAEIDFDKAVQ</entry></row><row><entry></entry></row><row><entry>TYGIAEGVGHGSLIGLVCAFHLSGFRLFSKAGEAMAFRNRSRYPTDAFAE</entry></row><row><entry></entry></row><row><entry>KLSAIMGIQLPTLSPEGLDLIFQSPPRSRDGIAPVWSENEVRNRLYTNWT</entry></row><row><entry></entry></row><row><entry>GRGPANKPDEHLLEIAGEIAKQVFPKFGGWDDLASDPDKALAAADKYFQS</entry></row><row><entry></entry></row><row><entry>QGDFPSIASLPAAIMLSPANSTVDFEGDYIAIDPAAETLLHQAVSRCAAR</entry></row><row><entry></entry></row><row><entry>LGRERPDLDQNKGPFVSSLQDALVSSQNNGLSWLFGVGFQHWKEKSPKEL</entry></row><row><entry></entry></row><row><entry>IDEYKVPADQHGAVTQVKSFVDAIPLNPLFDTTHYGEFRASVAGKVRSWV</entry></row><row><entry></entry></row><row><entry>ANYWKRLLDLKSLLATTEFTLPESISDPKAVSLFSGLLVDPQGLKKVADS</entry></row><row><entry></entry></row><row><entry>LPARLVSAEEAIDRLMGVGIPTAADIAQVERVADEIGAFIGQVQQFNNQV</entry></row><row><entry></entry></row><row><entry>KQKLENLQDADDEEFLKGLKIELPSGDKEPPAINRISGGAPDAAAEISEL</entry></row><row><entry></entry></row><row><entry>EEKLQRLLDARSEHFQTISEWAEENAVTLDPIAAMVELERLRLAERGATG</entry></row><row><entry></entry></row><row><entry>DPEEYALRLLLQRIGRLANRVSPVSAGSIRELLKPVFMEEREFNLFFHNR</entry></row><row><entry></entry></row><row><entry>LGSLYRSPYSTSRHQPFSIDVGKAKAIDWIAGLDQISSDIEKALSGAGEA</entry></row><row><entry></entry></row><row><entry>LGDQLRDWINLAGFAISQRLRGLPDTVPNALAQVRCPDDVRIPPLLAMLL</entry></row><row><entry></entry></row><row><entry>EEDDIARDVCLKAFNLYVSAINGCLFGALREGFIVRTRFQRIGTDQIHYV</entry></row><row><entry></entry></row><row><entry>PKDKAWEYPDRLNTAKGPINAAVSSDWIEKDGAVIKPVETVRNLSSTGFA</entry></row><row><entry></entry></row><row><entry>GAGVSEYLVQAPHDWYTPLDLRDVAHLVTGLPVEKNITKLKRLTNRTAFR</entry></row><row><entry></entry></row><row><entry>MVGASSFKTHLDSVLLSDKIKLGDFTIIIDQHYRQSVTYGGKVKISYEPE</entry></row><row><entry></entry></row><row><entry>RLQVEAAVPVVDTRDRTVPEPDTLFDHIVAIDLGERSVGFAVFDIKSCLR</entry></row><row><entry></entry></row><row><entry>TGEVKPIHDNNGNPVVGTVAVPSIRRLMKAVRSHRRRRQPNQKVNQTYST</entry></row><row><entry></entry></row><row><entry>ALQNYRENVIGDVCNRIDTLMERYNAFPVLEFQIKNFQAGAKQLEIVYGS</entry></row></tbody></tgroup></table></tables>

0297In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein is a Cas9 from archaea (e.g. nanoarchaea), which constitute a domain and kingdom of single-celled prokaryotic microbes. In some embodiments, the napDNAbp is CasX or CasY, which have been described in, for example, Burstein et al., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb. 21. doi: 10.1038/cr.2017.21, which is incorporated herein by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. In some embodiments, Cas9 refers to CasX, or a variant of CasX. In some embodiments, Cas9 refers to a CasY, or a variant of CasY. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp) and are within the scope of this disclosure.

0298In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein is a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp is a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 29-31. In some embodiments, the napDNAbp comprises an amino acid sequence of any one SEQ ID NOs: 29-31. It should be appreciated that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.

0000CasX (uniprot.org/uniprot/F0NN87; uniprot.org/uniprot/F0NH53) >tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx protein OS═<i>Sulfolobus islandicus </i>(strain HVE10/4) GN=SiH_0402 PE=4 SV=1

0299<tables id="TABLE-US-00028" num="00028"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 29)</entry></row><row><entry>MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAK</entry></row><row><entry></entry></row><row><entry>NNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFP</entry></row><row><entry></entry></row><row><entry>TTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE</entry></row><row><entry></entry></row><row><entry>VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNG</entry></row><row><entry></entry></row><row><entry>IVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGG</entry></row><row><entry></entry></row><row><entry>FSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG</entry></row><row><entry></entry></row><row><entry>SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG</entry></row></tbody></tgroup></table></tables><br /> >tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS═<i>Sulfolobus islandicus </i>(strain REY15A) GN=SiRe_0771 PE=4 SV=1

0300<tables id="TABLE-US-00029" num="00029"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 30)</entry></row><row><entry>MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAK</entry></row><row><entry></entry></row><row><entry>NNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFP</entry></row><row><entry></entry></row><row><entry>TTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLE</entry></row><row><entry></entry></row><row><entry>VEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNG</entry></row><row><entry></entry></row><row><entry>IVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGG</entry></row><row><entry></entry></row><row><entry>FSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG</entry></row><row><entry></entry></row><row><entry>SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG</entry></row></tbody></tgroup></table></tables>

0301CasY (ncbi.nlm.nih.gov/protein/APG80656.1)

0000>APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacterium]

0302<tables id="TABLE-US-00030" num="00030"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 31)</entry></row><row><entry>MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPRE</entry></row><row><entry></entry></row><row><entry>IVSAINDDYVGLYGLSNFDDLYNAEKRNEEKVYSVLDFWYDCVQYGAVFS</entry></row><row><entry></entry></row><row><entry>YTAPGLLKNVAEVRGGSYELTKTLKGSHLYDELQIDKVIKFLNKKEISRA</entry></row><row><entry></entry></row><row><entry>NGSLDKLKKDIIDCFKAEYRERHKDQCNKLADDIKNAKKDAGASLGERQK</entry></row><row><entry></entry></row><row><entry>KLFRDFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNRNRGEVLFNKL</entry></row><row><entry></entry></row><row><entry>KEYAQKLDKNEGSLEMWEYIGIGNSGTAFSNFLGEGFLGRLRENKITELK</entry></row><row><entry></entry></row><row><entry>KAMMDITDAWRGQEQEEELEKRLRILAALTIKLREPKFDNHWGGYRSDIN</entry></row><row><entry></entry></row><row><entry>GKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEMINRFGESDTKEEAVVS</entry></row><row><entry></entry></row><row><entry>SLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDVQE</entry></row><row><entry></entry></row><row><entry>ALIKERLEAEKKKKPKKRKKKSDAEDEKETIDFKELFPHLAKPLKLVPNF</entry></row><row><entry></entry></row><row><entry>YGDSKRELYKKYKNAAIYTDALWKAVEKIYKSAFSSSLKNSFFDTDFDKD</entry></row><row><entry></entry></row><row><entry>FFIKRLQKIFSVYRRFNTDKWKPIVKNSFAPYCDIVSLAENEVLYKPKQS</entry></row><row><entry></entry></row><row><entry>RSRKSAAIDKNRVRLPSTENIAKAGIALARELSVAGFDWKDLLKKEEHEE</entry></row><row><entry></entry></row><row><entry>YIDLIELHKTALALLLAVTETQLDISALDFVENGTVKDFMKTRDGNLVLE</entry></row><row><entry></entry></row><row><entry>GRFLEMFSQSIVFSELRGLAGLMSRKEFITRSAIQTMNGKQAELLYIPHE</entry></row><row><entry></entry></row><row><entry>FQSAKITTPKEMSRAFLDLAPAEFATSLEPESLSEKSLLKLKQMRYYPHY</entry></row><row><entry></entry></row><row><entry>FGYELTRTGQGIDGGVAENALRLEKSPVKKREIKCKQYKTLGRGQNKIVL</entry></row><row><entry></entry></row><row><entry>YVRSSYYQTQFLEWFLHRPKNVQTDVAVSGSFLIDEKKVKTRWNYDALTV</entry></row><row><entry></entry></row><row><entry>ALEPVSGSERVFVSQPFTIFPEKSAEEEGQRYLGIDIGEYGIAYTALEIT</entry></row><row><entry></entry></row><row><entry>GDSAKILDQNFISDPQLKTLREEVKGLKLDQRRGTFAMPSTKIARIRESL</entry></row><row><entry></entry></row><row><entry>VHSLRNRIHHLALKHKAKIVYELEVSRFEEGKQKIKKVYATLKKADVYSE</entry></row><row><entry></entry></row><row><entry>IDADKNLQTTVWGKLAVASEISASYTSQFCGACKKLWRAEMQVDETITTQ</entry></row><row><entry></entry></row><row><entry>ELIGTVRVIKGGTLIDAIKDFMRPPIFDENDTPFPKYRDFCDKHHISKKM</entry></row><row><entry></entry></row><row><entry>RGNSCLFICPFCRANADADIQASQTIALLRYVKEEKKVEDYFERFRKLKN</entry></row><row><entry></entry></row><row><entry>IKVLGQMKKI</entry></row></tbody></tgroup></table></tables><br /> Cas9 Domains of Nucleobase Editors

0303Non-limiting, exemplary Cas9 domains are provided herein. The Cas9 domain may be a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase. In some embodiments, the Cas9 domain is a nuclease active domain. For example, the Cas9 domain may be a Cas9 domain that cuts both strands of a duplexed nucleic acid (e.g., both strands of a duplexed DNA molecule). In some embodiments, the Cas9 domain comprises any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that has at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues as compared to any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein.

0304In some embodiments, the Cas9 domain is a nuclease-inactive Cas9 domain (dCas9). For example, the dCas9 domain may bind to a duplexed nucleic acid molecule (e.g., via a gRNA molecule) without cleaving either strand of the duplexed nucleic acid molecule. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10X mutation and a H840X mutation of the amino acid sequence set forth in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, wherein X is any amino acid change. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10A mutation and a H840A mutation of the amino acid sequence set forth in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. As one example, a nuclease-inactive Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 32 (Cloning vector pPlatTET-gRNA2, Accession No. BAV54124).

0305<tables id="TABLE-US-00031" num="00031"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG</entry></row><row><entry></entry></row><row><entry>ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFF</entry></row><row><entry></entry></row><row><entry>HRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD</entry></row><row><entry></entry></row><row><entry>KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF</entry></row><row><entry></entry></row><row><entry>EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS</entry></row><row><entry></entry></row><row><entry>LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAK</entry></row><row><entry></entry></row><row><entry>NLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL</entry></row><row><entry></entry></row><row><entry>PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK</entry></row><row><entry></entry></row><row><entry>LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE</entry></row><row><entry></entry></row><row><entry>KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS</entry></row><row><entry></entry></row><row><entry>FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAF</entry></row><row><entry></entry></row><row><entry>LSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFN</entry></row><row><entry></entry></row><row><entry>ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLK</entry></row><row><entry></entry></row><row><entry>TYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD</entry></row><row><entry></entry></row><row><entry>GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK</entry></row><row><entry></entry></row><row><entry>GILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRI</entry></row><row><entry></entry></row><row><entry>EEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRL</entry></row><row><entry></entry></row><row><entry>SDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY</entry></row><row><entry></entry></row><row><entry>WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV</entry></row><row><entry></entry></row><row><entry>AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN</entry></row><row><entry></entry></row><row><entry>YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI</entry></row><row><entry></entry></row><row><entry>GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGR</entry></row><row><entry></entry></row><row><entry>DFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD</entry></row><row><entry></entry></row><row><entry>PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEK</entry></row><row><entry></entry></row><row><entry>NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE</entry></row><row><entry></entry></row><row><entry>LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS</entry></row><row><entry></entry></row><row><entry>EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAA</entry></row><row><entry></entry></row><row><entry>FKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD </entry></row><row><entry></entry></row><row><entry>(SEQ ID NO: 32; see, e.g., Qi et al., Repurposing </entry></row><row><entry>CRISPR as an RNA-guided platform for sequence-</entry></row><row><entry>specific control of gene expression. Cell. 2013; </entry></row><row><entry>152(5):1173-83, the entire contents of which are </entry></row><row><entry>incorporated herein by reference).</entry></row></tbody></tgroup></table></tables>

0306Additional suitable nuclease-inactive dCas9 domains will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A/H840A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains (See, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. <i>Nature Biotechnology. </i>2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference). In some embodiments the dCas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the dCas9 domains provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequences that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more or more mutations compared to any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that has at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues as compared to any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein.

0307In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase may be a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule (e.g., a duplexed DNA molecule). In some embodiments the Cas9 nickase cleaves the target strand of a duplexed nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is base paired to (complementary to) a gRNA (e.g., an sgRNA) that is bound to the Cas9. In some embodiments, a Cas9 nickase comprises a D10A mutation and has a histidine at position 840 of SEQ ID NO: 6, or a mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. For example, a Cas9 nickase may comprise the amino acid sequence as set forth in SEQ ID NO: 8. In some embodiments the Cas9 nickase cleaves the non-target, non-base-edited strand of a duplexed nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is not base paired to a gRNA (e.g., an sgRNA) that is bound to the Cas9. In some embodiments, a Cas9 nickase comprises an H840A mutation and has an aspartic acid residue at position 10 of SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 nickases provided herein. Additional suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure.

0000Cas9 Domains with Reduced PAM Exclusivity

0308Some aspects of the disclosure provide Cas9 domains that have different PAM specificities. Typically, Cas9 proteins, such as Cas9 from <i>S. pyogenes </i>(spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region. This may limit the ability to edit desired bases within a genome. In some embodiments, the base editing fusion proteins provided herein may need to be placed at a precise location, for example where a target base is placed within a 4 base region (e.g., a “deamination window”), which is approximately 15 bases upstream of the PAM. See Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” <i>Nature </i>533, 420-424 (2016), the entire contents of which are hereby incorporated by reference. Accordingly, in some embodiments, any of the fusion proteins provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” <i>Nature </i>523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of <i>Staphylococcus aureus </i>CRISPR-Cas9 by modifying PAM recognition” <i>Nature Biotechnology </i>33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference.

0309In some embodiments, the Cas9 domain is a Cas9 domain from <i>Staphylococcus aureus </i>(SaCas9). In some embodiments, the SaCas9 domain is a nuclease active SaCas9, a nuclease inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, the SaCas9 comprises the amino acid sequence SEQ ID NO: 33. In some embodiments, the SaCas9 comprises a N579X mutation of SEQ ID NO: 33, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, wherein X is any amino acid except for N. In some embodiments, the SaCas9 comprises a N579A mutation of SEQ ID NO: 33, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a NNGRRT PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of a E781X, a N967X, and a R1014X mutation of SEQ ID NO: 33, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, wherein X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of a E781K, a N967K, and a R1014H mutation of SEQ ID NO: 33, or one or more corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the SaCas9 domain comprises a E781K, a N967K, or a R1014H mutation of SEQ ID NO: 33, or corresponding mutations in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein.

0310In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 33-36. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises the amino acid sequence of any one of SEQ ID NOs: 33-36. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein consists of the amino acid sequence of any one of SEQ ID NOs: 33-36.

0000Exemplary SaCas9 Sequence

0311<tables id="TABLE-US-00032" num="00032"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 33)</entry></row><row><entry>KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR</entry></row><row><entry></entry></row><row><entry>GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS</entry></row><row><entry></entry></row><row><entry>EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA</entry></row><row><entry></entry></row><row><entry>ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY</entry></row><row><entry></entry></row><row><entry>IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY</entry></row><row><entry></entry></row><row><entry>NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK</entry></row><row><entry></entry></row><row><entry>EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI</entry></row><row><entry></entry></row><row><entry>AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN</entry></row><row><entry></entry></row><row><entry>LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK</entry></row><row><entry></entry></row><row><entry>RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT</entry></row><row><entry></entry></row><row><entry>NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF</entry></row><row><entry></entry></row><row><entry>NYEVDHIIPRSVSFDNSFNNKVLVKQEE<u style="single"><b>N</b></u>SKKGNRTPFQYLSSSDSKISY</entry></row><row><entry></entry></row><row><entry>ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY</entry></row><row><entry></entry></row><row><entry>ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH</entry></row><row><entry></entry></row><row><entry>AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK</entry></row><row><entry></entry></row><row><entry>EIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI</entry></row><row><entry></entry></row><row><entry>VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK</entry></row><row><entry></entry></row><row><entry>NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR</entry></row><row><entry></entry></row><row><entry>NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK</entry></row><row><entry></entry></row><row><entry>KLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITY</entry></row><row><entry></entry></row><row><entry>REYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK</entry></row><row><entry></entry></row><row><entry>KG</entry></row></tbody></tgroup></table></tables><br /> Residue N579 of SEQ ID NO: 33, which is underlined and in bold, may be mutated (e.g., to a A579) to yield a SaCas9 nickase. <br /> Exemplary SaCas9d Sequence

0312<tables id="TABLE-US-00033" num="00033"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 34)</entry></row><row><entry>KRNYILGL<u style="single"><b>A</b></u>IGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR</entry></row><row><entry></entry></row><row><entry>GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS</entry></row><row><entry></entry></row><row><entry>EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA</entry></row><row><entry></entry></row><row><entry>ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY</entry></row><row><entry></entry></row><row><entry>IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY</entry></row><row><entry></entry></row><row><entry>NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK</entry></row><row><entry></entry></row><row><entry>EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI</entry></row><row><entry></entry></row><row><entry>AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN</entry></row><row><entry></entry></row><row><entry>LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK</entry></row><row><entry></entry></row><row><entry>RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT</entry></row><row><entry></entry></row><row><entry>NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF</entry></row><row><entry></entry></row><row><entry>NYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISY</entry></row><row><entry></entry></row><row><entry>ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY</entry></row><row><entry></entry></row><row><entry>ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH</entry></row><row><entry></entry></row><row><entry>AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK</entry></row><row><entry></entry></row><row><entry>EIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI</entry></row><row><entry></entry></row><row><entry>VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK</entry></row><row><entry></entry></row><row><entry>NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR</entry></row><row><entry></entry></row><row><entry>NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK</entry></row><row><entry></entry></row><row><entry>KLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITY</entry></row><row><entry></entry></row><row><entry>REYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK</entry></row><row><entry></entry></row><row><entry>KG</entry></row></tbody></tgroup></table></tables><br /> Residue D10 of SEQ ID NO: 34, which is underlined and in bold, may be mutated (e.g., to a A10) to yield a nuclease inactive SaCas9d. <br /> Exemplary SaCas9n Sequence

0313<tables id="TABLE-US-00034" num="00034"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 35)</entry></row><row><entry>KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR</entry></row><row><entry></entry></row><row><entry>GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS</entry></row><row><entry></entry></row><row><entry>EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA</entry></row><row><entry></entry></row><row><entry>ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY</entry></row><row><entry></entry></row><row><entry>IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY</entry></row><row><entry></entry></row><row><entry>NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK</entry></row><row><entry></entry></row><row><entry>EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI</entry></row><row><entry></entry></row><row><entry>AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN</entry></row><row><entry></entry></row><row><entry>LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK</entry></row><row><entry></entry></row><row><entry>RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT</entry></row><row><entry></entry></row><row><entry>NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF</entry></row><row><entry></entry></row><row><entry>NYEVDHIIPRSVSFDNSFNNKVLVKQEE<u style="single"><b>A</b></u>SKKGNRTPFQYLSSSDSKISY</entry></row><row><entry></entry></row><row><entry>ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY</entry></row><row><entry></entry></row><row><entry>ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH</entry></row><row><entry></entry></row><row><entry>AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK</entry></row><row><entry></entry></row><row><entry>EIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI</entry></row><row><entry></entry></row><row><entry>VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK</entry></row><row><entry></entry></row><row><entry>NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR</entry></row><row><entry></entry></row><row><entry>NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK</entry></row><row><entry></entry></row><row><entry>KLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITY</entry></row><row><entry></entry></row><row><entry>REYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK</entry></row><row><entry></entry></row><row><entry>KG.</entry></row></tbody></tgroup></table></tables><br /> Residue A579 of SEQ ID NO: 35, which can be mutated from N579 of SEQ ID NO: 33 to yield a SaCas9 nickase, is underlined and in bold. <br /> Exemplary SaKKH Cas9

0314<tables id="TABLE-US-00035" num="00035"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 36)</entry></row><row><entry>KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR</entry></row><row><entry></entry></row><row><entry>GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS</entry></row><row><entry></entry></row><row><entry>EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA</entry></row><row><entry></entry></row><row><entry>ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY</entry></row><row><entry></entry></row><row><entry>IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY</entry></row><row><entry></entry></row><row><entry>NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK</entry></row><row><entry></entry></row><row><entry>EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI</entry></row><row><entry></entry></row><row><entry>AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN</entry></row><row><entry></entry></row><row><entry>LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK</entry></row><row><entry></entry></row><row><entry>RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT</entry></row><row><entry></entry></row><row><entry>NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF</entry></row><row><entry></entry></row><row><entry>NYEVDHIIPRSVSFDNSFNNKVLVKQEE<u style="single"><b>A</b></u>SKKGNRTPFQYLSSSDSKISY</entry></row><row><entry></entry></row><row><entry>ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY</entry></row><row><entry></entry></row><row><entry>ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH</entry></row><row><entry></entry></row><row><entry>AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK</entry></row><row><entry></entry></row><row><entry>EIFITPHQIKHIKDFKDYKYSHRVDKKPNR<u style="single"><i>K</i></u>LINDTLYSTRKDDKGNTLI</entry></row><row><entry></entry></row><row><entry>VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK</entry></row><row><entry></entry></row><row><entry>NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR</entry></row><row><entry></entry></row><row><entry>NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK</entry></row><row><entry></entry></row><row><entry>KLKKISNQAEFIASFY<u style="single"><i>K</i></u>NDLIKINGELYRVIGVNNDLLNRIEVNMIDITY</entry></row><row><entry></entry></row><row><entry>REYLENMNDKRPP<u style="single"><i>H</i></u>IIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK</entry></row><row><entry></entry></row><row><entry>KG.</entry></row></tbody></tgroup></table></tables><br /> Residue A579 of SEQ ID NO: 36, which can be mutated from N579 of SEQ ID NO: 36 to yield a SaCas9 nickase, is underlined and in bold. Residues K781, K967, and H1014 of SEQ ID NO: 36, which can be mutated from E781, N967, and R1014 of SEQ ID NO: 36 to yield a SaKKH Cas9 are underlined and in italics.

0315In some embodiments, the Cas9 domain is a Cas9 domain from <i>Streptococcus pyogenes </i>(SpCas9). In some embodiments, the SpCas9 domain is a nuclease active SpCas9, a nuclease inactive SpCas9 (SpCas9d), or a SpCas9 nickase (SpCas9n). In some embodiments, the SpCas9 comprises the amino acid sequence SEQ ID NO: 37. In some embodiments, the SpCas9 comprises a D9X mutation of SEQ ID NO: 37, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, wherein X is any amino acid except for D. In some embodiments, the SpCas9 comprises a D9A mutation of SEQ ID NO: 37, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence having a NGG, a NGA, or a NGCG PAM sequence. In some embodiments, the SpCas9 domain comprises one or more of a D1134X, a R1334X, and a T1336X mutation of SEQ ID NO: 37, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1134E, R1334Q, and T1336R mutation of SEQ ID NO: 37, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the SpCas9 domain comprises a D1134E, a R1334Q, and a T1336R mutation of SEQ ID NO: 37, or corresponding mutations in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the SpCas9 domain comprises one or more of a D1134X, a R1334X, and a T1336X mutation of SEQ ID NO: 37, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1134V, a R1334Q, and a T1336R mutation of SEQ ID NO: 37, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the SpCas9 domain comprises a D1134V, a R1334Q, and a T1336R mutation of SEQ ID NO: 37, or corresponding mutations in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the SpCas9 domain comprises one or more of a D1134X, a G1217X, a R1334X, and a T1336X mutation of SEQ ID NO: 37, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1134V, a G1217R, a R1334Q, and a T1336R mutation of SEQ ID NO: 37, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the SpCas9 domain comprises a D1134V, a G1217R, a R1334Q, and a T1336R mutation of SEQ ID NO: 37, or corresponding mutations in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein.

0316In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 37-41. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises the amino acid sequence of any one of SEQ ID NOs: 37-41. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein consists of the amino acid sequence of any one of SEQ ID NOs: 37-41.

0000Exemplary SpCas9

0317<tables id="TABLE-US-00036" num="00036"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 37)</entry></row><row><entry>DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL</entry></row><row><entry></entry></row><row><entry>LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL</entry></row><row><entry></entry></row><row><entry>EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL</entry></row><row><entry></entry></row><row><entry>RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI</entry></row><row><entry></entry></row><row><entry>NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN</entry></row><row><entry></entry></row><row><entry>FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL</entry></row><row><entry></entry></row><row><entry>LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF</entry></row><row><entry></entry></row><row><entry>FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK</entry></row><row><entry></entry></row><row><entry>QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY</entry></row><row><entry></entry></row><row><entry>VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN</entry></row><row><entry></entry></row><row><entry>LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL</entry></row><row><entry></entry></row><row><entry>LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII</entry></row><row><entry></entry></row><row><entry>KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL</entry></row><row><entry></entry></row><row><entry>KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS</entry></row><row><entry></entry></row><row><entry>LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM</entry></row><row><entry></entry></row><row><entry>GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV</entry></row><row><entry></entry></row><row><entry>ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS</entry></row><row><entry></entry></row><row><entry>IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT</entry></row><row><entry></entry></row><row><entry>KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR</entry></row><row><entry></entry></row><row><entry>EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY</entry></row><row><entry></entry></row><row><entry>PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT</entry></row><row><entry></entry></row><row><entry>LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ</entry></row><row><entry></entry></row><row><entry>TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK</entry></row><row><entry></entry></row><row><entry>GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY</entry></row><row><entry></entry></row><row><entry>SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED</entry></row><row><entry></entry></row><row><entry>NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP</entry></row><row><entry></entry></row><row><entry>IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS</entry></row><row><entry></entry></row><row><entry>ITGLYETRIDLSQLGGD</entry></row></tbody></tgroup></table></tables><br /> Exemplary SpCas9n

0318<tables id="TABLE-US-00037" num="00037"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 38)</entry></row><row><entry>DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL</entry></row><row><entry></entry></row><row><entry>LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL</entry></row><row><entry></entry></row><row><entry>EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL</entry></row><row><entry></entry></row><row><entry>RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI</entry></row><row><entry></entry></row><row><entry>NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN</entry></row><row><entry></entry></row><row><entry>FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL</entry></row><row><entry></entry></row><row><entry>LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF</entry></row><row><entry></entry></row><row><entry>FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK</entry></row><row><entry></entry></row><row><entry>QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY</entry></row><row><entry></entry></row><row><entry>VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN</entry></row><row><entry></entry></row><row><entry>LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL</entry></row><row><entry></entry></row><row><entry>LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII</entry></row><row><entry></entry></row><row><entry>KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL</entry></row><row><entry></entry></row><row><entry>KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS</entry></row><row><entry></entry></row><row><entry>LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM</entry></row><row><entry></entry></row><row><entry>GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV</entry></row><row><entry></entry></row><row><entry>ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS</entry></row><row><entry></entry></row><row><entry>IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT</entry></row><row><entry></entry></row><row><entry>KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR</entry></row><row><entry></entry></row><row><entry>EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY</entry></row><row><entry></entry></row><row><entry>PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT</entry></row><row><entry></entry></row><row><entry>LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ</entry></row><row><entry></entry></row><row><entry>TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK</entry></row><row><entry></entry></row><row><entry>GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY</entry></row><row><entry></entry></row><row><entry>SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED</entry></row><row><entry></entry></row><row><entry>NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP</entry></row><row><entry></entry></row><row><entry>IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS</entry></row><row><entry></entry></row><row><entry>ITGLYETRIDLSQLGGD</entry></row></tbody></tgroup></table></tables><br /> Exemplary SpEQR Cas9

0319<tables id="TABLE-US-00038" num="00038"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 39)</entry></row><row><entry>DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL</entry></row><row><entry></entry></row><row><entry>LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL</entry></row><row><entry></entry></row><row><entry>EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL</entry></row><row><entry></entry></row><row><entry>RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI</entry></row><row><entry></entry></row><row><entry>NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN</entry></row><row><entry></entry></row><row><entry>FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL</entry></row><row><entry></entry></row><row><entry>LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF</entry></row><row><entry></entry></row><row><entry>FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK</entry></row><row><entry></entry></row><row><entry>QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY</entry></row><row><entry></entry></row><row><entry>VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN</entry></row><row><entry></entry></row><row><entry>LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL</entry></row><row><entry></entry></row><row><entry>LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII</entry></row><row><entry></entry></row><row><entry>KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL</entry></row><row><entry></entry></row><row><entry>KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS</entry></row><row><entry></entry></row><row><entry>LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM</entry></row><row><entry></entry></row><row><entry>GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV</entry></row><row><entry></entry></row><row><entry>ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS</entry></row><row><entry></entry></row><row><entry>IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT</entry></row><row><entry></entry></row><row><entry>KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR</entry></row><row><entry></entry></row><row><entry>EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY</entry></row><row><entry></entry></row><row><entry>PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT</entry></row><row><entry></entry></row><row><entry>LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ</entry></row><row><entry></entry></row><row><entry>TGGFSKESILPKRNSDKLIARKKDWDPKKYGGF<u style="single"><b>E</b></u>SPTVAYSVLVVAKVEK</entry></row><row><entry></entry></row><row><entry>GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY</entry></row><row><entry></entry></row><row><entry>SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED</entry></row><row><entry></entry></row><row><entry>NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP</entry></row><row><entry></entry></row><row><entry>IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK<u style="single"><b>Q</b></u>Y<u style="single"><b>R</b></u>STKEVLDATLIHQS</entry></row><row><entry></entry></row><row><entry>ITGLYETRIDLSQLGGD</entry></row></tbody></tgroup></table></tables><br /> Residues E1134, Q1334, and R1336 of SEQ ID NO: 39, which can be mutated from D1134, R1334, and T1336 of SEQ ID NO: 39 to yield a SpEQR Cas9, are underlined and in bold. <br /> Exemplary SpVQR Cas9

0320<tables id="TABLE-US-00039" num="00039"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 40)</entry></row><row><entry>DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL</entry></row><row><entry></entry></row><row><entry>LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL</entry></row><row><entry></entry></row><row><entry>EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL</entry></row><row><entry></entry></row><row><entry>RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI</entry></row><row><entry></entry></row><row><entry>NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN</entry></row><row><entry></entry></row><row><entry>FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL</entry></row><row><entry></entry></row><row><entry>LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF</entry></row><row><entry></entry></row><row><entry>FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK</entry></row><row><entry></entry></row><row><entry>QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY</entry></row><row><entry></entry></row><row><entry>VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN</entry></row><row><entry></entry></row><row><entry>LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL</entry></row><row><entry></entry></row><row><entry>LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII</entry></row><row><entry></entry></row><row><entry>KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL</entry></row><row><entry></entry></row><row><entry>KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS</entry></row><row><entry></entry></row><row><entry>LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM</entry></row><row><entry></entry></row><row><entry>GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV</entry></row><row><entry></entry></row><row><entry>ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS</entry></row><row><entry></entry></row><row><entry>IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT</entry></row><row><entry></entry></row><row><entry>KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR</entry></row><row><entry></entry></row><row><entry>EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY</entry></row><row><entry></entry></row><row><entry>PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT</entry></row><row><entry></entry></row><row><entry>LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ</entry></row><row><entry></entry></row><row><entry>TGGFSKESILPKRNSDKLIARKKDWDPKKYGGF<u style="single"><b>V</b></u>SPTVAYSVLVVAKVEK</entry></row><row><entry></entry></row><row><entry>GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY</entry></row><row><entry></entry></row><row><entry>SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED</entry></row><row><entry></entry></row><row><entry>NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP</entry></row><row><entry></entry></row><row><entry>IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK<u style="single"><b>Q</b></u>Y<u style="single"><b>R</b></u>STKEVLDATLIHQS</entry></row><row><entry></entry></row><row><entry>ITGLYETRIDLSQLGGD</entry></row></tbody></tgroup></table></tables><br /> Residues V1134, Q1334, and R1336 of SEQ ID NO: 40, which can be mutated from D1134, R1334, and T1336 of SEQ ID NO: 40 to yield a SpVQR Cas9, are underlined and in bold. <br /> Exemplary SpVRER Cas9

0321<tables id="TABLE-US-00040" num="00040"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 41)</entry></row><row><entry>DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL</entry></row><row><entry></entry></row><row><entry>LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL</entry></row><row><entry></entry></row><row><entry>EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL</entry></row><row><entry></entry></row><row><entry>RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI</entry></row><row><entry></entry></row><row><entry>NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN</entry></row><row><entry></entry></row><row><entry>FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL</entry></row><row><entry></entry></row><row><entry>LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF</entry></row><row><entry></entry></row><row><entry>FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK</entry></row><row><entry></entry></row><row><entry>QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY</entry></row><row><entry></entry></row><row><entry>VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN</entry></row><row><entry></entry></row><row><entry>LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL</entry></row><row><entry></entry></row><row><entry>LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII</entry></row><row><entry></entry></row><row><entry>KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL</entry></row><row><entry></entry></row><row><entry>KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS</entry></row><row><entry></entry></row><row><entry>LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM</entry></row><row><entry></entry></row><row><entry>GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV</entry></row><row><entry></entry></row><row><entry>ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS</entry></row><row><entry></entry></row><row><entry>IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT</entry></row><row><entry></entry></row><row><entry>KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR</entry></row><row><entry></entry></row><row><entry>EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY</entry></row><row><entry></entry></row><row><entry>PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT</entry></row><row><entry></entry></row><row><entry>LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ</entry></row><row><entry></entry></row><row><entry>TGGFSKESILPKRNSDKLIARKKDWDPKKYGGF<u style="single"><b>V</b></u>SPTVAYSVLVVAKVEK</entry></row><row><entry></entry></row><row><entry>GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY</entry></row><row><entry></entry></row><row><entry>SLFELENGRKRMLASA<u style="single"><b>R</b></u>ELQKGNELALPSKYVNFLYLASHYEKLKGSPED</entry></row><row><entry></entry></row><row><entry>NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP</entry></row><row><entry></entry></row><row><entry>IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK<u style="single"><b>E</b></u>Y<u style="single"><b>R</b></u>STKEVLDATLIHQS</entry></row><row><entry></entry></row><row><entry>ITGLYETRIDLSQLGGD</entry></row></tbody></tgroup></table></tables><br /> Residues V1134, R1217, Q1334, and R1336 of SEQ ID NO: 41, which can be mutated from D1134, G1217, R1334, and T1336 of SEQ ID NO: 41 to yield a SpVRER Cas9, are underlined and in bold.

0322The following are exemplary fusion proteins (e.g., base editing proteins) capable of binding to a nucleic acid sequence having a non-canonical (e.g., a non-NGG) PAM sequence:

0000Exemplary SaBE3 (rAPOBEC1-XTEN-SaCas9n-UGI-NLS)

0323<tables id="TABLE-US-00041" num="00041"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 42)</entry></row><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPES<u style="single">KRNYI</u></entry></row><row><entry></entry></row><row><entry><u style="single">LGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRL</u></entry></row><row><entry></entry></row><row><entry><u style="single">KRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFS</u></entry></row><row><entry></entry></row><row><entry><u style="single">AALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLE</u></entry></row><row><entry></entry></row><row><entry><u style="single">RLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLE</u></entry></row><row><entry></entry></row><row><entry><u style="single">TRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLY</u></entry></row><row><entry></entry></row><row><entry><u style="single">NALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVN</u></entry></row><row><entry></entry></row><row><entry><u style="single">EEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILT</u></entry></row><row><entry></entry></row><row><entry><u style="single">IYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDE</u></entry></row><row><entry></entry></row><row><entry><u style="single">LWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQ</u></entry></row><row><entry></entry></row><row><entry><u style="single">SIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIE</u></entry></row><row><entry></entry></row><row><entry><u style="single">EIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVD</u></entry></row><row><entry></entry></row><row><entry><u style="single">HIIPRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKK</u></entry></row><row><entry></entry></row><row><entry><u style="single">HILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGL</u></entry></row><row><entry></entry></row><row><entry><u style="single">MNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDAL</u></entry></row><row><entry></entry></row><row><entry><u style="single">IIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFIT</u></entry></row><row><entry></entry></row><row><entry><u style="single">PHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLN</u></entry></row><row><entry></entry></row><row><entry><u style="single">GLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYK</u></entry></row><row><entry></entry></row><row><entry><u style="single">YYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVK</u></entry></row><row><entry></entry></row><row><entry><u style="single">LSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKI</u></entry></row><row><entry></entry></row><row><entry><u style="single">SNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLE</u></entry></row><row><entry></entry></row><row><entry><u style="single">NMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG</u>SGG</entry></row><row><entry></entry></row><row><entry>STNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDES</entry></row><row><entry></entry></row><row><entry>TDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV</entry></row></tbody></tgroup></table></tables><br /> Exemplary SaKKH-BE3 (rAPOBEC1-XTEN-SaCas9n-UGI-NLS)

0324<tables id="TABLE-US-00042" num="00042"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 43)</entry></row><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPES<u style="single">KRNYI</u></entry></row><row><entry></entry></row><row><entry><u style="single">LGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRL</u></entry></row><row><entry></entry></row><row><entry><u style="single">KRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFS</u></entry></row><row><entry></entry></row><row><entry><u style="single">AALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLE</u></entry></row><row><entry></entry></row><row><entry><u style="single">RLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLE</u></entry></row><row><entry></entry></row><row><entry><u style="single">TRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLY</u></entry></row><row><entry></entry></row><row><entry><u style="single">NALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVN</u></entry></row><row><entry></entry></row><row><entry><u style="single">EEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILT</u></entry></row><row><entry></entry></row><row><entry><u style="single">IYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDE</u></entry></row><row><entry></entry></row><row><entry><u style="single">LWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQ</u></entry></row><row><entry></entry></row><row><entry><u style="single">SIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIE</u></entry></row><row><entry></entry></row><row><entry><u style="single">EIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVD</u></entry></row><row><entry></entry></row><row><entry><u style="single">HIIPRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKK</u></entry></row><row><entry></entry></row><row><entry><u style="single">HILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGL</u></entry></row><row><entry></entry></row><row><entry><u style="single">MNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDAL</u></entry></row><row><entry></entry></row><row><entry><u style="single">IIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFIT</u></entry></row><row><entry></entry></row><row><entry><u style="single">PHQIKHIKDFKDYKYSHRVDKKPNR<i>K</i>LINDTLYSTRKDDKGNTLIVNNLN</u></entry></row><row><entry></entry></row><row><entry><u style="single">GLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYK</u></entry></row><row><entry></entry></row><row><entry><u style="single">YYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVK</u></entry></row><row><entry></entry></row><row><entry><u style="single">LSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKI</u></entry></row><row><entry></entry></row><row><entry><u style="single">SNQAEFIASFY<i>K</i>NDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLE</u></entry></row><row><entry></entry></row><row><entry><u style="single">NMNDKRPP<i>H</i>IIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG</u>SGG</entry></row><row><entry></entry></row><row><entry>STNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDES</entry></row><row><entry></entry></row><row><entry>TDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV</entry></row></tbody></tgroup></table></tables><br /> Exemplary EQR-BE3 (rAPOBEC1-XTEN-Cas9n-UGI-NLS)

0325<tables id="TABLE-US-00043" num="00043"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 44)</entry></row><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPES<u style="single">DKKYS</u></entry></row><row><entry></entry></row><row><entry><u style="single">IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG</u></entry></row><row><entry></entry></row><row><entry><u style="single">ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL</u></entry></row><row><entry></entry></row><row><entry><u style="single">VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL</u></entry></row><row><entry></entry></row><row><entry><u style="single">ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV</u></entry></row><row><entry></entry></row><row><entry><u style="single">DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF</u></entry></row><row><entry></entry></row><row><entry><u style="single">DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL</u></entry></row><row><entry></entry></row><row><entry><u style="single">RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK</u></entry></row><row><entry></entry></row><row><entry><u style="single">NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD</u></entry></row><row><entry></entry></row><row><entry><u style="single">NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA</u></entry></row><row><entry></entry></row><row><entry><u style="single">RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK</u></entry></row><row><entry></entry></row><row><entry><u style="single">VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN</u></entry></row><row><entry></entry></row><row><entry><u style="single">RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF</u></entry></row><row><entry></entry></row><row><entry><u style="single">LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY</u></entry></row><row><entry></entry></row><row><entry><u style="single">TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE</u></entry></row><row><entry></entry></row><row><entry>DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP</entry></row><row><entry></entry></row><row><entry><u style="single">ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL</u></entry></row><row><entry></entry></row><row><entry><u style="single">QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV</u></entry></row><row><entry></entry></row><row><entry><u style="single">LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG</u></entry></row><row><entry></entry></row><row><entry><u style="single">GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI</u></entry></row><row><entry></entry></row><row><entry><u style="single">TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES</u></entry></row><row><entry></entry></row><row><entry><u style="single">EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE</u></entry></row><row><entry></entry></row><row><entry><u style="single">IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS</u></entry></row><row><entry></entry></row><row><entry><u style="single">KESILPKRNSDKLIARKKDWDPKKYGGF<i>E</i>SPTVAYSVLVVAKVEKGKSKK</u></entry></row><row><entry></entry></row><row><entry><u style="single">LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL</u></entry></row><row><entry></entry></row><row><entry><u style="single">ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ</u></entry></row><row><entry></entry></row><row><entry><u style="single">LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA</u></entry></row><row><entry></entry></row><row><entry><u style="single">ENIIHLFTLTNLGAPAAFKYFDTTIDRK<i>Q</i>Y<i>R</i>STKEVLDATLIHQSITGLY</u></entry></row><row><entry></entry></row><row><entry><u style="single">ETRIDLSQLGGD</u>SGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGN</entry></row><row><entry></entry></row><row><entry>KPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLS</entry></row><row><entry></entry></row><row><entry>GGSPKKKRKV</entry></row></tbody></tgroup></table></tables><br /> VQR-BE3 (rAPOBEC1-XTEN-Cas9n-UGI-NLS)

0326<tables id="TABLE-US-00044" num="00044"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 45)</entry></row><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPES<u style="single">DKKYS</u></entry></row><row><entry></entry></row><row><entry><u style="single">IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG</u></entry></row><row><entry></entry></row><row><entry><u style="single">ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL</u></entry></row><row><entry></entry></row><row><entry><u style="single">VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL</u></entry></row><row><entry></entry></row><row><entry><u style="single">ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV</u></entry></row><row><entry></entry></row><row><entry><u style="single">DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF</u></entry></row><row><entry></entry></row><row><entry><u style="single">DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL</u></entry></row><row><entry></entry></row><row><entry><u style="single">RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK</u></entry></row><row><entry></entry></row><row><entry><u style="single">NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD</u></entry></row><row><entry></entry></row><row><entry><u style="single">NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA</u></entry></row><row><entry></entry></row><row><entry><u style="single">RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK</u></entry></row><row><entry></entry></row><row><entry><u style="single">VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN</u></entry></row><row><entry></entry></row><row><entry><u style="single">RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF</u></entry></row><row><entry></entry></row><row><entry><u style="single">LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY</u></entry></row><row><entry></entry></row><row><entry><u style="single">TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE</u></entry></row><row><entry></entry></row><row><entry><u style="single">DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP</u></entry></row><row><entry></entry></row><row><entry><u style="single">ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL</u></entry></row><row><entry></entry></row><row><entry><u style="single">QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV</u></entry></row><row><entry></entry></row><row><entry><u style="single">LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG</u></entry></row><row><entry></entry></row><row><entry><u style="single">GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI</u></entry></row><row><entry></entry></row><row><entry><u style="single">TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES</u></entry></row><row><entry></entry></row><row><entry><u style="single">EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE</u></entry></row><row><entry></entry></row><row><entry><u style="single">IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS</u></entry></row><row><entry></entry></row><row><entry><u style="single">KESILPKRNSDKLIARKKDWDPKKYGGF<i>V</i>SPTVAYSVLVVAKVEKGKSKK</u></entry></row><row><entry></entry></row><row><entry><u style="single">LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL</u></entry></row><row><entry></entry></row><row><entry><u style="single">ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ</u></entry></row><row><entry></entry></row><row><entry><u style="single">LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA</u></entry></row><row><entry></entry></row><row><entry><u style="single">ENIIHLFTLTNLGAPAAFKYFDTTIDRK<i>Q</i>Y<i>R</i>STKEVLDATLIHQSITGLY</u></entry></row><row><entry></entry></row><row><entry><u style="single">ETRIDLSQLGGD</u>SGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGN</entry></row><row><entry></entry></row><row><entry>KPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLS</entry></row><row><entry></entry></row><row><entry>GGSPKKKRKV</entry></row></tbody></tgroup></table></tables><br /> VRER-BE3 (rAPOBEC1-XTEN-Cas9n-UGI-NLS)

0327<tables id="TABLE-US-00045" num="00045"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 46)</entry></row><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPES<u style="single">DKKYS</u></entry></row><row><entry></entry></row><row><entry><u style="single">IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG</u></entry></row><row><entry></entry></row><row><entry><u style="single">ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL</u></entry></row><row><entry></entry></row><row><entry><u style="single">VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL</u></entry></row><row><entry></entry></row><row><entry><u style="single">ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV</u></entry></row><row><entry></entry></row><row><entry><u style="single">DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF</u></entry></row><row><entry></entry></row><row><entry><u style="single">DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL</u></entry></row><row><entry></entry></row><row><entry><u style="single">RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK</u></entry></row><row><entry></entry></row><row><entry><u style="single">NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD</u></entry></row><row><entry></entry></row><row><entry><u style="single">NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA</u></entry></row><row><entry></entry></row><row><entry><u style="single">RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK</u></entry></row><row><entry></entry></row><row><entry><u style="single">VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN</u></entry></row><row><entry></entry></row><row><entry><u style="single">RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF</u></entry></row><row><entry></entry></row><row><entry><u style="single">LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY</u></entry></row><row><entry></entry></row><row><entry><u style="single">TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE</u></entry></row><row><entry></entry></row><row><entry><u style="single">DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP</u></entry></row><row><entry></entry></row><row><entry><u style="single">ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL</u></entry></row><row><entry></entry></row><row><entry><u style="single">QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV</u></entry></row><row><entry></entry></row><row><entry><u style="single">LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG</u></entry></row><row><entry></entry></row><row><entry><u style="single">GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI</u></entry></row><row><entry></entry></row><row><entry><u style="single">TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES</u></entry></row><row><entry></entry></row><row><entry><u style="single">EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE</u></entry></row><row><entry></entry></row><row><entry><u style="single">IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS</u></entry></row><row><entry></entry></row><row><entry><u style="single">KESILPKRNSDKLIARKKDWDPKKYGGF<i>V</i>SPTVAYSVLVVAKVEKGKSKK</u></entry></row><row><entry></entry></row><row><entry><u style="single">LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL</u></entry></row><row><entry></entry></row><row><entry><u style="single">ENGRKRMLASA<i>R</i>ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ</u></entry></row><row><entry></entry></row><row><entry><u style="single">LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA</u></entry></row><row><entry></entry></row><row><entry><u style="single">ENIIHLFTLTNLGAPAAFKYFDTTIDRK<i>E</i>Y<i>R</i>STKEVLDATLIHQSITGLY</u></entry></row><row><entry></entry></row><row><entry><u style="single">ETRIDLSQLGGD</u>SGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGN</entry></row><row><entry></entry></row><row><entry>KPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLS</entry></row><row><entry></entry></row><row><entry>GGSPKKKRKV</entry></row></tbody></tgroup></table></tables><br /> High Fidelity Base Editors

0328Some aspects of the disclosure provide Cas9 fusion proteins (e.g., any of the fusion proteins provided herein) comprising a Cas9 domain that has high fidelity. Additional aspects of the disclosure provide Cas9 fusion proteins (e.g., any of the fusion proteins provided herein) comprising a Cas9 domain with decreased electrostatic interactions between the Cas9 domain and a sugar-phosphate backbone of a DNA, as compared to a wild-type Cas9 domain. In some embodiments, a Cas9 domain (e.g., a wild type Cas9 domain) comprises one or more mutations that decreases the association between the Cas9 domain and a sugar-phosphate backbone of a DNA. In some embodiments, any of the Cas9 fusion proteins provided herein comprise one or more of a N497X, a R661X, a Q695X, and/or a Q926X mutation of the amino acid sequence provided in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, wherein X is any amino acid. In some embodiments, any of the Cas9 fusion proteins provided herein comprise one or more of a N497A, a R661A, a Q695A, and/or a Q926A mutation of the amino acid sequence provided in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the Cas9 domain comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the Cas9 domain (e.g., of any of the fusion proteins provided herein) comprises the amino acid sequence as set forth in SEQ ID NO: 47. In some embodiments, the fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 48. Cas9 domains with high fidelity are known in the art and would be apparent to the skilled artisan. For example, Cas9 domains with high fidelity have been described in Kleinstiver, B. P., et al. “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.” <i>Nature </i>529, 490-495 (2016); and Slaymaker, I. M., et al. “Rationally engineered Cas9 nucleases with improved specificity.” <i>Science </i>351, 84-88 (2015); the entire contents of each are incorporated herein by reference.

0329It should be appreciated that the base editors provided herein, for example, base editor 2 (BE2) or base editor 3 (BE3), may be converted into high fidelity base editors by modifying the Cas9 domain as described herein to generate high fidelity base editors, for example, high fidelity base editor 2 (HF-BE2) or high fidelity base editor 3 (HF-BE3). In some embodiments, base editor 2 (BE2) comprises a deaminase domain, a dCas9, and a UGI domain. In some embodiments, base editor 3 (BE3) comprises a deaminase domain, anCas9 domain and a UGI domain.

0000Cas9 Domain where Mutations Relative to Cas9 of SEQ ID NO: 6 are Shown in Bold and Underlines

0330<tables id="TABLE-US-00046" num="00046"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 47)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>DKKYSIGL<u style="single"><b>A</b></u>IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL</entry></row><row><entry></entry></row><row><entry>LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL</entry></row><row><entry></entry></row><row><entry>EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL</entry></row><row><entry></entry></row><row><entry>RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI</entry></row><row><entry></entry></row><row><entry>NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN</entry></row><row><entry></entry></row><row><entry>FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL</entry></row><row><entry></entry></row><row><entry>LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF</entry></row><row><entry></entry></row><row><entry>FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK</entry></row><row><entry></entry></row><row><entry>QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY</entry></row><row><entry></entry></row><row><entry>VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT<u style="single"><b>A</b></u>FDKN</entry></row><row><entry></entry></row><row><entry>LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL</entry></row><row><entry></entry></row><row><entry>LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII</entry></row><row><entry></entry></row><row><entry>KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL</entry></row><row><entry></entry></row><row><entry>KRRRYTGWG<u style="single"><b>A</b></u>LSRKLINGIRDKQSGKTILDFLKSDGFANRNFM<u style="single"><b>A</b></u>LIHDDS</entry></row><row><entry></entry></row><row><entry>LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM</entry></row><row><entry></entry></row><row><entry>GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV</entry></row><row><entry></entry></row><row><entry>ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS</entry></row><row><entry></entry></row><row><entry>IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT</entry></row><row><entry></entry></row><row><entry>KAERGGLSELDKAGFIKRQLVETR<u style="single"><b>A</b></u>ITKHVAQILDSRMNTKYDENDKLIR</entry></row><row><entry></entry></row><row><entry>EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY</entry></row><row><entry></entry></row><row><entry>PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT</entry></row><row><entry></entry></row><row><entry>LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ</entry></row><row><entry></entry></row><row><entry>TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK</entry></row><row><entry></entry></row><row><entry>GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY</entry></row><row><entry></entry></row><row><entry>SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED</entry></row><row><entry></entry></row><row><entry>NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP</entry></row><row><entry></entry></row><row><entry>IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS</entry></row><row><entry></entry></row><row><entry>ITGLYETRIDLSQLGGD</entry></row></tbody></tgroup></table></tables><br /> HF-BE3

0331<tables id="TABLE-US-00047" num="00047"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 48)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYS</entry></row><row><entry></entry></row><row><entry>IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG</entry></row><row><entry></entry></row><row><entry>ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL</entry></row><row><entry></entry></row><row><entry>VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL</entry></row><row><entry></entry></row><row><entry>ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV</entry></row><row><entry></entry></row><row><entry>DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF</entry></row><row><entry></entry></row><row><entry>DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL</entry></row><row><entry></entry></row><row><entry>RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK</entry></row><row><entry></entry></row><row><entry>NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD</entry></row><row><entry></entry></row><row><entry>NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA</entry></row><row><entry></entry></row><row><entry>RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEK</entry></row><row><entry></entry></row><row><entry>VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN</entry></row><row><entry></entry></row><row><entry>RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF</entry></row><row><entry></entry></row><row><entry>LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY</entry></row><row><entry></entry></row><row><entry>TGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMALIHDDSLTFKE</entry></row><row><entry></entry></row><row><entry>DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP</entry></row><row><entry></entry></row><row><entry>ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL</entry></row><row><entry></entry></row><row><entry>QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV</entry></row><row><entry></entry></row><row><entry>LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG</entry></row><row><entry></entry></row><row><entry>GLSELDKAGFIKRQLVETRAITKHVAQILDSRMNTKYDENDKLIREVKVI</entry></row><row><entry></entry></row><row><entry>TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES</entry></row><row><entry></entry></row><row><entry>EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE</entry></row><row><entry></entry></row><row><entry>IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS</entry></row><row><entry></entry></row><row><entry>KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK</entry></row><row><entry></entry></row><row><entry>LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL</entry></row><row><entry></entry></row><row><entry>ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ</entry></row><row><entry></entry></row><row><entry>LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA</entry></row><row><entry></entry></row><row><entry>ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY</entry></row><row><entry></entry></row><row><entry>ETRIDLSQLGGD</entry></row></tbody></tgroup></table></tables><br /> Cas9 Fusion Proteins

0332Any of the Cas9 domains (e.g., a nuclease active Cas9 protein, a nuclease-inactive dCas9 protein, or a Cas9 nickase protein) disclosed herein may be fused to a second protein, thus fusion proteins provided herein comprise a Cas9 domain as provided herein and a second protein, or a “fusion partner”. In some embodiments, the second protein is fused to the N-terminus of the Cas9 domain. However, in other embodiments, the second protein is fused to the C-terminus of the Cas9 domain. In some embodiments, the second protein that is fused to the Cas9 domain is a nucleic acid editing domain. In some embodiments, the Cas9 domain and the nucleic acid editing domain are fused via a linker, while in other embodiments the Cas9 domain and the nucleic acid editing domain are fused directly to one another. In some embodiments, the Cas9 domain and the nucleic acid editing domain are fused via a linker of any length or composition. For example, the linker may be a bond, one or more amino acids, a peptide, or a polymer, of any length and composition. In some embodiments, the linker comprises (GGGS)_n(SEQ ID NO: 613), (GGGGS)_n(SEQ ID NO: 607), (G)_n(SEQ ID NO: 608), (EAAAK)_n(SEQ ID NO: 609), (GGS)_n(SEQ ID NO: 610), (SGGS)_n(SEQ ID NO: 606), SGSETPGTSESATPES (SEQ ID NO: 604), SGGS(GGS)_n(SEQ ID NO: 612), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605), or (XP)_n(SEQ ID NO: 611) motif, or a combination of any of these, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, the linker comprises a (GGS)_nmotif, wherein n is 1, 3, or 7. In some embodiments, the linker comprises a (GGS)_n(SEQ ID NO: 610) motif, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises the amino acid sequence SGGS(GGS)_n(SEQ ID NO: 612), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker comprises the amino acid sequence SGGS(GGS)_n(SEQ ID NO: 612), wherein n is 2. In some embodiments, the linker comprises an amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 604), also referred to as the XTEN linker in the Examples). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605), also referred to as the 32 amino acid linker in the Examples. The length of the linker can influence the base to be edited, as illustrated in the Examples. For example, a linker of 3-amino-acid long (e.g., (GGS)₁) may give a 2-5, 2-4, 2-3, 3-4 base editing window relative to the PAM sequence, while a 9-amino-acid linker (e.g., (GGS)₃(SEQ ID NO: 610)) may give a 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, 5-6 base editing window relative to the PAM sequence. A 16-amino-acid linker (e.g., the XTEN linker) may give a 2-7, 2-6, 2-5, 2-4, 2-3, 3-7, 3-6, 3-5, 3-4, 4-7, 4-6, 4-5, 5-7, 5-6, 6-7 base window relative to the PAM sequence with exceptionally strong activity, and a 21-amino-acid linker (e.g., (GGS)₇(SEQ ID NO: 610)) may give a 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, 7-8 base editing window relative to the PAM sequence. The novel finding that varying linker length may allow the dCas9 fusion proteins of the disclosure to edit nucleobases different distances from the PAM sequence affords significant clinical importance, since a PAM sequence may be of varying distance to the disease-causing mutation to be corrected in a gene. It is to be understood that the linker lengths described as examples here are not meant to be limiting.

0333In some embodiments, the second protein comprises an enzymatic domain. In some embodiments, the enzymatic domain is a nucleic acid editing domain. Such a nucleic acid editing domain may be, without limitation, a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, or an acetyltransferase. Non-limiting exemplary binding domains that may be used in accordance with this disclosure include transcriptional activator domains and transcriptional repressor domains.

0000Deaminase Domains

0334In some embodiments, second protein comprises a nucleic acid editing domain. In some embodiments, the nucleic acid editing domain can catalyze a C to U base change. In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytidine deaminase or a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase. In some embodiments, the deaminase is an APOBEC2 deaminase. In some embodiments, the deaminase is an APOBEC3 deaminase. In some embodiments, the deaminase is an APOBEC3A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase. In some embodiments, the deaminase is an APOBEC3E deaminase. In some embodiments, the deaminase is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a vertebrate deaminase. In some embodiments, the deaminase is an invertebrate deaminase. In some embodiments, the deaminase is a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse deaminase. In some embodiments, the deaminase is a human deaminase. In some embodiments, the deaminase is a rat deaminase, e.g., rAPOBEC1. In some embodiments, the deaminase is an activation-induced cytidine deaminase (AID). In some embodiments, the deaminase is a cytidine deaminase 1 (CDA1). In some embodiments, the deaminase is a <i>Petromyzon marinus </i>cytidine deaminase 1 (pmCDA1). In some embodiments, the deminase is a human APOBEC3G (SEQ ID NO: 60). In some embodiments, the deaminase is a fragment of the human APOBEC3G (SEQ ID NO: 83). In some embodiments, the deaminase is a human APOBEC3G variant comprising a D316R_D317R mutation (SEQ ID NO: 82). In some embodiments, the deaminase is a fragment of the human APOBEC3G and comprising mutations corresponding to the D316R_D317R mutations in SEQ ID NO: 60 (SEQ ID NO: 84).

0335In some embodiments, the nucleic acid editing domain is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the deaminase domain of any one of SEQ ID NOs: 49-84. In some embodiments, the nucleic acid editing domain comprises the amino acid sequence of any one of SEQ ID NOs: 49-84.

0000Deaminase Domains that Modulate the Editing Window of Base Editors

0336Some aspects of the disclosure are based on the recognition that modulating the deaminase domain catalytic activity of any of the fusion proteins provided herein, for example by making point mutations in the deaminase domain, affect the processivity of the fusion proteins (e.g., base editors). For example, mutations that reduce, but do not eliminate, the catalytic activity of a deaminase domain within a base editing fusion protein can make it less likely that the deaminase domain will catalyze the deamination of a residue adjacent to a target residue, thereby narrowing the deamination window. The ability to narrow the deaminataion window may prevent unwanted deamination of residues adjacent of specific target residues, which may decrease or prevent off-target effects.

0337In some embodiments, any of the fusion proteins provided herein comprise a deaminase domain (e.g., a cytidine deaminase domain) that has reduced catalytic deaminase activity. In some embodiments, any of the fusion proteins provided herein comprise a deaminase domain (e.g., a cytidine deaminase domain) that has a reduced catalytic deaminase activity as compared to an appropriate control. For example, the appropriate control may be the deaminase activity of the deaminase prior to introducing one or more mutations into the deaminase. In other embodiments, the appropriate control may be a wild-type deaminase. In some embodiments, the appropriate control is a wild-type apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the appropriate control is an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, or an APOBEC3H deaminase. In some embodiments, the appropriate control is an activation induced deaminase (AID). In some embodiments, the appropriate control is a cytidine deaminase 1 from <i>Petromyzon marinus </i>(pmCDA1). In some embodiments, the deaminase domain may be a deaminase domain that has at least 1%, at least 5%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% less catalytic deaminase activity as compared to an appropriate control.

0338In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising one or more mutations selected from the group consisting of H121X, H122X, R126X, R126X, R118X, W90X, W90X, and R132X of rAPOBEC1 (SEQ ID NO: 76), or one or more corresponding mutations in another APOBEC deaminase, wherein X is any amino acid. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising one or more mutations selected from the group consisting of H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBEC1 (SEQ ID NO: 76), or one or more corresponding mutations in another APOBEC deaminase.

0339In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising one or more mutations selected from the group consisting of D316X, D317X, R320X, R320X, R313X, W285X, W285X, R326X of hAPOBEC3G (SEQ ID NO: 60), or one or more corresponding mutations in another APOBEC deaminase, wherein X is any amino acid. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising one or more mutations selected from the group consisting of D316R, D317R, R320A, R320E, R313A, W285A, W285Y, R326E of hAPOBEC3G (SEQ ID NO: 60), or one or more corresponding mutations in another APOBEC deaminase.

0340In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a H121R and a H122R mutation of rAPOBEC1 (SEQ ID NO: 76), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R126A mutation of rAPOBEC1 (SEQ ID NO: 76), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R126E mutation of rAPOBEC1 (SEQ ID NO: 76), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R118A mutation of rAPOBEC1 (SEQ ID NO: 76), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W90A mutation of rAPOBEC1 (SEQ ID NO: 76), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W90Y mutation of rAPOBEC1 (SEQ ID NO: 76), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R132E mutation of rAPOBEC1 (SEQ ID NO: 76), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W90Y and a R126E mutation of rAPOBEC1 (SEQ ID NO: 76), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R126E and a R132E mutation of rAPOBEC1 (SEQ ID NO: 76), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W90Y and a R132E mutation of rAPOBEC1 (SEQ ID NO: 76), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W90Y, R126E, and R132E mutation of rAPOBEC1 (SEQ ID NO: 76), or one or more corresponding mutations in another APOBEC deaminase.

0341In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a D316R and a D317R mutation of hAPOBEC3G (SEQ ID NO: 60), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R320A mutation of hAPOBEC3G (SEQ ID NO: 60), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R320E mutation of hAPOBEC3G (SEQ ID NO: 60), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R313A mutation of hAPOBEC3G (SEQ ID NO: 60), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W285A mutation of hAPOBEC3G (SEQ ID NO: 60), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W285Y mutation of hAPOBEC3G (SEQ ID NO: 60), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R326E mutation of hAPOBEC3G (SEQ ID NO: 60), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W285Y and a R320E mutation of hAPOBEC3G (SEQ ID NO: 60), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R320E and a R326E mutation of hAPOBEC3G (SEQ ID NO: 60), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W285Y and a R326E mutation of hAPOBEC3G (SEQ ID NO: 60), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W285Y, R320E, and R326E mutation of hAPOBEC3G (SEQ ID NO: 60), or one or more corresponding mutations in another APOBEC deaminase.

0342Some aspects of this disclosure provide fusion proteins comprising (i) a nuclease-inactive Cas9 domain; and (ii) a nucleic acid editing domain. In some embodiments, a nuclease-inactive Cas9 domain (dCas9), comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 as provided by any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, and comprises mutations that inactivate the nuclease activity of Cas9. Mutations that render the nuclease domains of Cas9 inactive are well-known in the art. For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of <i>S. pyogenes </i>Cas9 (Jinek et al., <i>Science. </i>337:816-821(2012); Qi et al., <i>Cell. </i>28; 152(5):1173-83 (2013)). In some embodiments, the dCas9 of this disclosure comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the dCas9 of this disclosure comprises a H840A mutation of the amino acid sequence provided in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the dCas9 of this disclosure comprises both D10A and H840A mutations of the amino acid sequence provided in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the Cas9 further comprises a histidine residue at position 840 of the amino acid sequence provided in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. The presence of the catalytic residue H840 restores the activity of the Cas9 to cleave the non-edited strand containing a G opposite the targeted C. Restoration of H840 does not result in the cleavage of the target strand containing the C. In some embodiments, the dCas9 comprises an amino acid sequence of SEQ ID NO: 32. It is to be understood that other mutations that inactivate the nuclease domains of Cas9 may also be included in the dCas9 of this disclosure.

0343The Cas9 or dCas9 domains comprising the mutations disclosed herein, may be a full-length Cas9, or a fragment thereof. In some embodiments, proteins comprising Cas9, or fragments thereof, are referred to as “Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to wild type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9, e.g., a Cas9 comprising the amino acid sequence of SEQ ID NO: 6.

0344Any of the Cas9 fusion proteins of this disclosure may further comprise a nucleic acid editing domain (e.g., an enzyme that is capable of modifying nucleic acid, such as a deaminase). In some embodiments, the nucleic acid editing domain is a DNA-editing domain. In some embodiments, the nucleic acid editing domain has deaminase activity. In some embodiments, the nucleic acid editing domain comprises or consists of a deaminase or deaminase domain. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 family deaminase. In some embodiments, the deaminase is an activation-induced cytidine deaminase (AID). Some nucleic-acid editing domains as well as Cas9 fusion proteins including such domains are described in detail herein. Additional suitable nucleic acid editing domains will be apparent to the skilled artisan based on this disclosure and knowledge in the field.

0345Some aspects of the disclosure provide a fusion protein comprising a Cas9 domain fused to a nucleic acid editing domain, wherein the nucleic acid editing domain is fused to the N-terminus of the Cas9 domain. In some embodiments, the Cas9 domain and the nucleic acid editing-editing domain are fused via a linker. In some embodiments, the linker comprises a (GGGS)_n(SEQ ID NO: 613), a (GGGGS)_n(SEQ ID NO: 607), a (G)_n(SEQ ID NO: 608), an (EAAAK)_n(SEQ ID NO: 609), a (GGS)_n(SEQ ID NO: 610), (SGGS)_n(SEQ ID NO: 606), an SGSETPGTSESATPES (SEQ ID NO: 604) motif (see, e.g., Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. <i>Nat. Biotechnol. </i>2014; 32(6): 577-82; the entire contents are incorporated herein by reference), or an (XP)_n(SEQ ID NO: 611) motif, or a combination of any of these, wherein n is independently an integer between 1 and 30. In some embodiments, n is independently 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or, if more than one linker or more than one linker motif is present, any combination thereof. In some embodiments, the linker comprises a (GGS)_n(SEQ ID NO: 610) motif, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In some embodiments, the linker comprises a (GGS)_n(SEQ ID NO: 610) motif, wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604). Additional suitable linker motifs and linker configurations will be apparent to those of skill in the art. In some embodiments, suitable linker motifs and configurations include those described in Chen et al., Fusion protein linkers: property, design and functionality. <i>Adv Drug Deliv Rev. </i>2013; 65(10):1357-69, the entire contents of which are incorporated herein by reference. Additional suitable linker sequences will be apparent to those of skill in the art based on the instant disclosure. In some embodiments, the general architecture of exemplary Cas9 fusion proteins provided herein comprises the structure: <br />[NH₂]-[nucleic acid editing domain]-[Cas9]-[COOH] or<br />[NH₂]-[nucleic acid editing domain]-[linker]-[Cas9]-[COOH],<br /> wherein NH₂is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein.

0346The fusion proteins of the present disclosure may comprise one or more additional features. For example, in some embodiments, the fusion protein comprises a nuclear localization sequence (NLS). In some embodiments, the NLS of the fusion protein is localized between the nucleic acid editing domain and the Cas9 domain. In some embodiments, the NLS of the fusion protein is localized C-terminal to the Cas9 domain.

0347Other exemplary features that may be present are localization sequences, such as cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein comprises one or more His tags.

0348In some embodiments, the nucleic acid editing domain is a deaminase. For example, in some embodiments, the general architecture of exemplary Cas9 fusion proteins with a deaminase domain comprises the structure: <br />[NH₂]-[NLS]-[deaminase]-[Cas9]-[COOH],<br />[NH₂]-[Cas9]-[deaminase]-[COOH],<br />[NH₂]-[deaminase]-[Cas9]-[COOH], or<br />[NH₂]-[deaminase]-[Cas9]-[NLS]-[COOH];<br /> wherein NLS is a nuclear localization sequence, NH₂is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., PCT/EP2000/011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, a NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 614) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 615). In some embodiments, a linker is inserted between the Cas9 and the deaminase. In some embodiments, the NLS is located C-terminal of the Cas9 domain. In some embodiments, the NLS is located N-terminal of the Cas9 domain. In some embodiments, the NLS is located between the deaminase and the Cas9 domain. In some embodiments, the NLS is located N-terminal of the deaminase domain. In some embodiments, the NLS is located C-terminal of the deaminase domain.

0349One exemplary suitable type of nucleic acid editing domain is a cytidine deaminase, for example, of the APOBEC family. The apolipoprotein B mRNA-editing complex (APOBEC) family of cytidine deaminase enzymes encompasses eleven proteins that serve to initiate mutagenesis in a controlled and beneficial manner.²⁹One family member, activation-induced cytidine deaminase (AID), is responsible for the maturation of antibodies by converting cytosines in ssDNA to uracils in a transcription-dependent, strand-biased fashion.³⁰The apolipoprotein B editing complex 3 (APOBEC3) enzyme provides protection to human cells against a certain HIV-1 strain via the deamination of cytosines in reverse-transcribed viral ssDNA.³¹These proteins all require a Zn²⁺-coordinating motif (His-X-Glu-X_23-26-Pro-Cys-X_2-4-Cys; SEQ ID NO: 616) and bound water molecule for catalytic activity. The Glu residue acts to activate the water molecule to a zinc hydroxide for nucleophilic attack in the deamination reaction. Each family member preferentially deaminates at its own particular “hotspot”, ranging from WRC (W is A or T, R is A or G) for hAID, to TTC for hAPOBEC3F.³²A recent crystal structure of the catalytic domain of APOBEC3G revealed a secondary structure comprised of a five-stranded j-sheet core flanked by six a-helices, which is believed to be conserved across the entire family.³³The active center loops have been shown to be responsible for both ssDNA binding and in determining “hotspot” identity.³⁴Overexpression of these enzymes has been linked to genomic instability and cancer, thus highlighting the importance of sequence-specific targeting.³⁵

0350Some aspects of this disclosure relate to the recognition that the activity of cytidine deaminase enzymes such as APOBEC enzymes can be directed to a specific site in genomic DNA. Without wishing to be bound by any particular theory, advantages of using Cas9 as a recognition agent include (1) the sequence specificity of Cas9 can be easily altered by simply changing the sgRNA sequence; and (2) Cas9 binds to its target sequence by denaturing the dsDNA, resulting in a stretch of DNA that is single-stranded and therefore a viable substrate for the deaminase. It should be understood that other catalytic domains, or catalytic domains from other deaminases, can also be used to generate fusion proteins with Cas9, and that the disclosure is not limited in this regard.

0351Some aspects of this disclosure are based on the recognition that Cas9:deaminase fusion proteins can efficiently deaminate nucleotides at positions 3-11 according to the numbering scheme in <figref idref="DRAWINGS">FIG. 3</figref>. In view of the results provided herein regarding the nucleotides that can be targeted by Cas9:deaminase fusion proteins, a person of skill in the art will be able to design suitable guide RNAs to target the fusion proteins to a target sequence that comprises a nucleotide to be deaminated.

0352In some embodiments, the deaminase domain and the Cas9 domain are fused to each other via a linker. Various linker lengths and flexibilities between the deaminase domain (e.g., AID) and the Cas9 domain can be employed (e.g., ranging from very flexible linkers of the form (GGGGS)_n(SEQ ID NO: 607), (GGS)_n(SEQ ID NO: 610), and (G)_n(SEQ ID NO: 608) to more rigid linkers of the form (EAAAK)_n(SEQ ID NO: 609), (SGGS)_n(SEQ ID NO: 606), SGSETPGTSESATPES (SEQ ID NO: 604) (see, e.g., Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. <i>Nat. Biotechnol. </i>2014; 32(6): 577-82; the entire contents are incorporated herein by reference) and (XP)_n(SEQ ID NO: 611))³⁶in order to achieve the optimal length for deaminase activity for the specific application. In some embodiments, the linker comprises a (GGS)_n(SEQ ID NO: 610) motif, wherein n is 1, 3, or 7. In some embodiments, the linker comprises a (an SGSETPGTSESATPES (SEQ ID NO: 604) motif.

0353Some exemplary suitable nucleic-acid editing domains, e.g., deaminases and deaminase domains, that can be fused to Cas9 domains according to aspects of this disclosure are provided below. It should be understood that, in some embodiments, the active domain of the respective sequence can be used, e.g., the domain without a localizing signal (nuclear localization sequence, without nuclear export signal, cytoplasmic localizing signal).

0354Human AID:

0355<tables id="TABLE-US-00048" num="00048"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 49)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry><u style="single">MDSLLMNRRKFLYQFKNVRWAKGRRETYLC</u>YVVKRRDSATSFSLDFGYLR</entry></row><row><entry></entry></row><row><entry>NKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG</entry></row><row><entry></entry></row><row><entry>NPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNT</entry></row><row><entry></entry></row><row><entry>FVENHERTFKAWEGLHENSVRLSRQLRRILLP<u style="double">LYEVDDLRDAFRTLGL</u></entry></row></tbody></tgroup></table></tables><br /> (underline: nuclear localization sequence; double underline: nuclear export signal)

0356Mouse AID:

0357<tables id="TABLE-US-00049" num="00049"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 51)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry><u style="single">MDSLLMKQKKFLYHFKNVRWAKGRHETYLC</u>YVVKRRDSATSCSLDFGHLR</entry></row><row><entry></entry></row><row><entry>NKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVAEFLRW</entry></row><row><entry></entry></row><row><entry>NPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIGIMTFKDYFYCWNT</entry></row><row><entry></entry></row><row><entry>FVENRERTFKAWEGLHENSVRLTRQLRRILLP<u style="double">LYEVDDLRDAFRMLGF</u></entry></row></tbody></tgroup></table></tables><br /> (underline: nuclear localization sequence; double underline: nuclear export signal)

0358Dog AID:

0359<tables id="TABLE-US-00050" num="00050"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 52)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry><u style="single">MDSLLMKQRKFLYHFKNVRWAKGRHETYLC</u>YVVKRRDSATSFSLDFGHLR</entry></row><row><entry></entry></row><row><entry>NKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG</entry></row><row><entry></entry></row><row><entry>YPNLSLRIFAARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNT</entry></row><row><entry></entry></row><row><entry>FVENREKTFKAWEGLHENSVRLSRQLRRILLP<u style="double">LYEVDDLRDAFRTLGL</u></entry></row></tbody></tgroup></table></tables><br /> (underline: nuclear localization sequence; double underline: nuclear export signal)

0360Bovine AID:

0361<tables id="TABLE-US-00051" num="00051"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 53)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry><u style="single">MDSLLKKQRQFLYQFKNVRWAKGRHETYLC</u>YVVKRRDSPTSFSLDFGHLR</entry></row><row><entry></entry></row><row><entry>NKAGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG</entry></row><row><entry></entry></row><row><entry>YPNLSLRIFTARLYFCDKERKAEPEGLRRLHRAGVQIAIMTFKDYFYCWN</entry></row><row><entry></entry></row><row><entry>TFVENHERTFKAWEGLHENSVRLSRQLRRILLP<u style="double">LYEVDDLRDAFRTLGL</u></entry></row></tbody></tgroup></table></tables><br /> (underline: nuclear localization sequence; double underline: nuclear export signal)

0362<tables id="TABLE-US-00052" num="00052"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 54)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>Rat:AID:<u style="single">MAVGSKPKAALVGPHWERERIWCFLC</u>STGLGTQQTGQTSRWL</entry></row><row><entry></entry></row><row><entry>RPAATQDPVSPPRSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSA</entry></row><row><entry></entry></row><row><entry>TSFSLDFGYLRNKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYD</entry></row><row><entry></entry></row><row><entry>CARHVADFLRGNPNLSLRIFTARLTGWGALPAGLMSPARPSDYFYCWNTF</entry></row><row><entry></entry></row><row><entry>V<u style="double">ENHERTFKAWEGLHENSVRLSRRLRRILLPLYEVDDLRDAFRTLGL</u></entry></row></tbody></tgroup></table></tables><br /> (underline: nuclear localization sequence; double underline: nuclear export signal)

0363Mouse APOBEC-3:

0364<tables id="TABLE-US-00053" num="00053"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 55)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTR</entry></row><row><entry></entry></row><row><entry>KDCDSPVSLHHGVFKNKDNI<i>HAEICFLYWFHDKVLKVLSPREEFKITWYM</i></entry></row><row><entry></entry></row><row><entry><i>SWSPCFEC</i>AEQIVRFLATHHNLSLDIFSSRLYNVQDPETQQNLCRLVQEG</entry></row><row><entry></entry></row><row><entry>AQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNFRYQDSKLQEILRPC</entry></row><row><entry></entry></row><row><entry>YIPVPSSSSSTLSNICLTKGLPETRFCVEGRRMDPLSEEEFYSQFYNQRV</entry></row><row><entry></entry></row><row><entry>KHLCYYHRMKPYLCYQLEQFNGQAPLKGCLLSEKGKQ<i>HAEILFLDKIRSM</i></entry></row><row><entry></entry></row><row><entry><i>ELSQVTITCYLTWSPCPNC</i>AWQLAAFKRDRPDLILHIYTSRLYFHWKRPF</entry></row><row><entry></entry></row><row><entry>QKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQ</entry></row><row><entry></entry></row><row><entry>RRLRRIKESWGLQDLVNDFGNLQLGPPMS</entry></row></tbody></tgroup></table></tables><br /> (italic: nucleic acid editing domain)

0365Rat APOBEC-3:

0366<tables id="TABLE-US-00054" num="00054"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 56)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLRYAIDRKDTFLCYEVTR</entry></row><row><entry></entry></row><row><entry>KDCDSPVSLHHGVFKNKDNI<i>HAEICFLYWFHDKVLKVLSPREEFKITWYM</i></entry></row><row><entry></entry></row><row><entry><i>SWSPCFEC</i>AEQVLRFLATHHNLSLDIFSSRLYNIRDPENQQNLCRLVQEG</entry></row><row><entry></entry></row><row><entry>AQVAAMDLYEFKKCWKKFVDNGGRRFRPWKKLLTNFRYQDSKLQEILRPC</entry></row><row><entry></entry></row><row><entry>YIPVPSSSSSTLSNICLTKGLPETRFCVERRRVHLLSEEEFYSQFYNQRV</entry></row><row><entry></entry></row><row><entry>KHLCYYHGVKPYLCYQLEQFNGQAPLKGCLLSEKGKQ<i>HAEILFLDKIRSM</i></entry></row><row><entry></entry></row><row><entry><i>ELSQVIITCYLTWSPCPNC</i>AWQLAAFKRDRPDLILHIYTSRLYFHWKRPF</entry></row><row><entry></entry></row><row><entry>QKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQ</entry></row><row><entry></entry></row><row><entry>RRLHRIKESWGLQDLVNDFGNLQLGPPMS</entry></row></tbody></tgroup></table></tables><br /> (italic: nucleic acid editing domain)

0367Rhesus macaque APOBEC-3G:

0368<tables id="TABLE-US-00055" num="00055"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 57)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry><u style="single">MVEPMDPRTFVSNFNNRPILSGLNTVWLCCEVKTKDPSGPPLDAKIFQGK</u></entry></row><row><entry></entry></row><row><entry><u style="single">VYSKAKY<i>HPEM</i></u><i>RFLRWFHKWRQLHHDQEYKVTWYVSWSPCTRC</i>ANSVATF</entry></row><row><entry></entry></row><row><entry>LAKDPKVTLTIFVARLYYFWKPDYQQALRILCQKRGGPHATMKIMNYNEF</entry></row><row><entry></entry></row><row><entry>QDCWNKFVDGRGKPFKPRNNLPKHYTLLQATLGELLRHLMDPGTFTSNFN</entry></row><row><entry></entry></row><row><entry>NKPWVSGQHETYLCYKVERLHNDTWVPLNQHRGFLRNQAPNIHGFPKGR<i>H</i></entry></row><row><entry></entry></row><row><entry><i>AELCFLDLIPFWKLDGQQYRVTCFTSWSPCFSC</i>AQEMAKFISNNEHVSLC</entry></row><row><entry></entry></row><row><entry>IFAARIYDDQGRYQEGLRALHRDGAKIAMMNYSEFEYCWDTFVDRQGRPF</entry></row><row><entry></entry></row><row><entry>QPWDGLDEHSQALSGRLRAI</entry></row></tbody></tgroup></table></tables><br /> (italic: nucleic acid editing domain; underline: cytoplasmic localization signal)

0369Chimpanzee APOBEC-3G:

0370<tables id="TABLE-US-00056" num="00056"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 58)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry><u style="single">MKPHFRNPVERMYQDTFSDNFYNRPILSHRNTVWLCYEVKTKGPSRPPLD</u></entry></row><row><entry></entry></row><row><entry><u style="single">AKIFRGQVYS</u>KLKY<i>HPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKC</i></entry></row><row><entry></entry></row><row><entry>TRDVATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMK</entry></row><row><entry></entry></row><row><entry>IMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPP</entry></row><row><entry></entry></row><row><entry>TFTSNFNNELWVRGRHETYLCYEVERLHNDTWVLLNQRRGFLCNQAPHKH</entry></row><row><entry></entry></row><row><entry>GFLEGR<i>HAELCFLDVIPFWKLDLHQDYRVTCFTSWSPCFSC</i>AQEMAKFIS</entry></row><row><entry></entry></row><row><entry>NNKHVSLCIFAARIYDDQGRCQEGLRTLAKAGAKISIMTYSEFKHCWDTF</entry></row><row><entry></entry></row><row><entry>VDHQGCPFQPWDGLEEHSQALSGRLRAILQNQGN</entry></row></tbody></tgroup></table></tables><br /> (italic: nucleic acid editing domain; underline: cytoplasmic localization signal)

0371Green Monkey APOBEC-3G:

0372<tables id="TABLE-US-00057" num="00057"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 59)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry><u style="single">MNPQIRNMVEQMEPDIFVYYFNNRPILSGRNTVWLCYEVKTKDPSGPPLD</u></entry></row><row><entry></entry></row><row><entry><u style="single">ANIFQGKLYP</u>EAKD<i>HPEMKFLHWFRKWRQLHRDQEYEVTWYVSWSPCTRC</i></entry></row><row><entry></entry></row><row><entry>ANSVATFLAEDPKVTLTIFVARLYYFWKPDYQQALRILCQERGGPHATMK</entry></row><row><entry></entry></row><row><entry>IMNYNEFQHCWNEFVDGQGKPFKPRKNLPKHYTLLHATLGELLRHVMDPG</entry></row><row><entry></entry></row><row><entry>TFTSNFNNKPWVSGQRETYLCYKVERSHNDTWVLLNQHRGFLRNQAPDRH</entry></row><row><entry></entry></row><row><entry>GFPKGR<i>HAELCFLDLIPFWKLDDQQYRVTCFTSWSPCFSC</i>AQKMAKFISN</entry></row><row><entry></entry></row><row><entry>NKHVSLCIFAARIYDDQGRCQEGLRTLHRDGAKIAVMNYSEFEYCWDTFV</entry></row><row><entry></entry></row><row><entry>DRQGRPFQPWDGLDEHSQALSGRLRAI</entry></row></tbody></tgroup></table></tables><br /> (italic: nucleic acid editing domain; underline: cytoplasmic localization signal)

0373Human APOBEC-3G:

0374<tables id="TABLE-US-00058" num="00058"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 60)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry><u style="single">MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLD</u></entry></row><row><entry></entry></row><row><entry><u style="single">AKIFRGQVYS</u>ELKY<i>HPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKC</i></entry></row><row><entry></entry></row><row><entry>TRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMK</entry></row><row><entry></entry></row><row><entry>IMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPP</entry></row><row><entry></entry></row><row><entry>TFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKH</entry></row><row><entry></entry></row><row><entry>GFLEGR<i>HAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSC</i>AQEMAKFIS</entry></row><row><entry></entry></row><row><entry>KNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTF</entry></row><row><entry></entry></row><row><entry>VDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN</entry></row></tbody></tgroup></table></tables><br /> (italic: nucleic acid editing domain; underline: cytoplasmic localization signal)

0375Human APOBEC-3F:

0376<tables id="TABLE-US-00059" num="00059"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 61)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLD</entry></row><row><entry></entry></row><row><entry>AKIFRGQVYSQPEH<i>HAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDC</i>V</entry></row><row><entry></entry></row><row><entry>AKLAEFLAEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVKIMDDE</entry></row><row><entry></entry></row><row><entry>EFAYCWENFVYSEGQPFMPWYKFDDNYAFLHRTLKEILRNPMEAMYPHIF</entry></row><row><entry></entry></row><row><entry>YFHFKNLRKAYGRNESWLCFTMEVVKHHSPVSWKRGVFRNQVDPETHC<i>HA</i></entry></row><row><entry></entry></row><row><entry><i>ERCFLSWFCDDILSPNTNYEVTWYTSWSPCPEC</i>AGEVAEFLARHSNVNLT</entry></row><row><entry></entry></row><row><entry>IFTARLYYFWDTDYQEGLRSLSQEGASVEIMGYKDFKYCWENFVYNDDEP</entry></row><row><entry></entry></row><row><entry>FKPWKGLKYNFLFLDSKLQEILE</entry></row></tbody></tgroup></table></tables><br /> (italic: nucleic acid editing domain)

0377Human APOBEC-3B:

0378<tables id="TABLE-US-00060" num="00060"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 62)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLW</entry></row><row><entry></entry></row><row><entry>DTGVFRGQVYFKPQY<i>HAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDC</i></entry></row><row><entry></entry></row><row><entry>VAKLAEFLSEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVTIMDY</entry></row><row><entry></entry></row><row><entry>EEFAYCWENFVYNEGQQFMPWYKFDENYAFLHRTLKEILRYLMDPDTFTF</entry></row><row><entry></entry></row><row><entry>NFNNDPLVLRRRQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFY</entry></row><row><entry></entry></row><row><entry><i>GRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGC</i>AGEVRAFLQEN</entry></row><row><entry></entry></row><row><entry>THVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVY</entry></row><row><entry></entry></row><row><entry>RQGCPFQPWDGLEEHSQALSGRLRAILQNQGN</entry></row></tbody></tgroup></table></tables><br /> (italic: nucleic acid editing domain)

0379Rat APOBEC-3B:

0380<tables id="TABLE-US-00061" num="00061"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 63)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MQPQGLGPNAGMGPVCLGCSHRRPYSPIRNPLKKLYQQTFYFHFKNVRYA</entry></row><row><entry></entry></row><row><entry>WGRKNNFLCYEVNGMDCALPVPLRQGVFRKQGHIHAELCFIYWFHDKVLR</entry></row><row><entry></entry></row><row><entry>VLSPMEEFKVTWYMSWSPCSKCAEQVARFLAAHRNLSLAIFSSRLYYYLR</entry></row><row><entry></entry></row><row><entry>NPNYQQKLCRLIQEGVHVAAMDLPEFKKCWNKFVDNDGQPFRPWMRLRIN</entry></row><row><entry></entry></row><row><entry>FSFYDCKLQEIFSRMNLLREDVFYLQFNNSHRVKPVQNRYYRRKSYLCYQ</entry></row><row><entry></entry></row><row><entry>LERANGQEPLKGYLLYKKGEQHVEILFLEKMRSMELSQVRITCYLTWSPC</entry></row><row><entry></entry></row><row><entry>PNCARQLAAFKKDHPDLILRIYTSRLYFYWRKKFQKGLCTLWRSGIHVDV</entry></row><row><entry></entry></row><row><entry>MDLPQFADCWTNFVNPQRPFRPWNELEKNSWRIQRRLRRIKESWGL</entry></row></tbody></tgroup></table></tables>

0381Bovine APOBEC-3B:

0382<tables id="TABLE-US-00062" num="00062"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 64)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>DGWEVAFRSGTVLKAGVLGVSMTEGWAGSGHPGQGACVWTPGTRNTMNLL</entry></row><row><entry></entry></row><row><entry>REVLFKQQFGNQPRVPAPYYRRKTYLCYQLKQRNDLTLDRGCFRNKKQRH</entry></row><row><entry></entry></row><row><entry>AEIRFIDKINSLDLNPSQSYKIICYITWSPCPNCANELVNFITRNNHLKL</entry></row><row><entry></entry></row><row><entry>EIFASRLYFHWIKSFKMGLQDLQNAGISVAVMTHTEFEDCWEQFVDNQSR</entry></row><row><entry></entry></row><row><entry>PFQPWDKLEQYSASIRRRLQRILTAPI</entry></row></tbody></tgroup></table></tables>

0383Chimpanzee APOBEC-3B:

0384<tables id="TABLE-US-00063" num="00063"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 65)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MNPQIRNPMEWMYQRTFYYNFENEPILYGRSYTWLCYEVKIRRGHSNLLW</entry></row><row><entry></entry></row><row><entry>DTGVFRGQMYSQPEHHAEMCFLSWFCGNQLSAYKCFQITWFVSWTPCPDC</entry></row><row><entry></entry></row><row><entry>VAKLAKFLAEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVKIMDD</entry></row><row><entry></entry></row><row><entry>EEFAYCWENFVYNEGQPFMPWYKFDDNYAFLHRTLKEIIRHLMDPDTFTF</entry></row><row><entry></entry></row><row><entry>NFNNDPLVLRRHQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFY</entry></row><row><entry></entry></row><row><entry>GRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGQVRAFLQEN</entry></row><row><entry></entry></row><row><entry>THVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVY</entry></row><row><entry></entry></row><row><entry>RQGCPFQPWDGLEEHSQALSGRLRAILQVRASSLCMVPHRPPPPPQSPGP</entry></row><row><entry></entry></row><row><entry>CLPLCSEPPLGSLLPTGRPAPSLPFLLTASFSFPPPASLPPLPSLSLSPG</entry></row><row><entry></entry></row><row><entry>HLPVPSFHSLTSCSIQPPCSSRIRETEGWASVSKEGRDLG</entry></row></tbody></tgroup></table></tables>

0385Human APOBEC-3C:

0386<tables id="TABLE-US-00064" num="00064"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 66)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSW</entry></row><row><entry></entry></row><row><entry>KTGVFRNQVDSETH<i>CHAERCFLSWFCDDILSPNTKYQVTWYTSWSPCPDC</i></entry></row><row><entry></entry></row><row><entry>AGEVAEFLARHSNVNLTIFTARLYYFQYPCYQEGLRSLSQEGVAVEIMDY</entry></row><row><entry></entry></row><row><entry>EDFKYCWENFVYNDNEPFKPWKGLKTNFRLLKRRLRESLQ</entry></row></tbody></tgroup></table></tables><br /> (italic: nucleic acid editing domain)

0387Gorilla APOBEC3C:

0388<tables id="TABLE-US-00065" num="00065"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 67)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSW</entry></row><row><entry></entry></row><row><entry>KTGVFRNQVDSETH<i>CHAERCFLSWFCDDILSPNTNYQVTWYTSWSPCPEC</i></entry></row><row><entry></entry></row><row><entry>AGEVAEFLARHSNVNLTIFTARLYYFQDTDYQEGLRSLSQEGVAVKIMDY</entry></row><row><entry></entry></row><row><entry>KDFKYCWENFVYNDDEPFKPWKGLKYNFRFLKRRLQEILE</entry></row></tbody></tgroup></table></tables><br /> (italic: nucleic acid editing domain)

0389Human APOBEC-3A:

0390<tables id="TABLE-US-00066" num="00066"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 68)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQ</entry></row><row><entry></entry></row><row><entry>HRGFLHNQAKNLLCGFYGR<i>HAELRFLDLVPSLQLDPAQIYRVTWFISWSP</i></entry></row><row><entry></entry></row><row><entry><i>CFSWGC</i>AGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQV</entry></row><row><entry></entry></row><row><entry>SIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN</entry></row></tbody></tgroup></table></tables><br /> (italic: nucleic acid editing domain)

0391Rhesus Macaque APOBEC-3A:

0392<tables id="TABLE-US-00067" num="00067"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 69)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MDGSPASRPRHLMDPNTFTFNFNNDLSVRGRHQTYLCYEVERLDNGTWVP</entry></row><row><entry></entry></row><row><entry>MDERRGFLCNKAKNVPCGDYGC<i>HVELRFLCEVPSWQLDPAQTYRVTWFIS</i></entry></row><row><entry></entry></row><row><entry><i>WSPC</i>FRRGCAGQVRVFLQENKHVRLRIFAARIYDYDPLYQEALRTLRDAG</entry></row><row><entry></entry></row><row><entry>AQVSIMTYEEFKHCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAILQNQ</entry></row><row><entry></entry></row><row><entry>GN</entry></row></tbody></tgroup></table></tables><br /> (italic: nucleic acid editing domain)

0393Bovine APOBEC-3A:

0394<tables id="TABLE-US-00068" num="00068"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 70)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MDEYTFTENFNNQGWPSKTYLCYEMERLDGDATIPLDEYKGFVRNKGLDQ</entry></row><row><entry></entry></row><row><entry>PEKPC<i>HAELYFLGKIHSWNLDRNQHYRLTCFISWSPC</i>YDCAQKLTTFLKE</entry></row><row><entry></entry></row><row><entry>NHHISLHILASRIYTHNRFGCHQSGLCELQAAGARITIMTFEDFKHCWET</entry></row><row><entry></entry></row><row><entry>FVDHKGKPFQPWEGLNVKSQALCTELQAILKTQQN</entry></row></tbody></tgroup></table></tables><br /> (italic: nucleic acid editing domain)

0395Human APOBEC-3H:

0396<tables id="TABLE-US-00069" num="00069"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 71)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENK</entry></row><row><entry></entry></row><row><entry>KKC<i>HAEICFINEIKSMGLDETQCYQVTCYLTWSPCSSC</i>AWELVDFIKAHD</entry></row><row><entry></entry></row><row><entry>HLNLGIFASRLYYHWCKPQQKGLRLLCGSQVPVEVMGFPKFADCWENFVD</entry></row><row><entry></entry></row><row><entry>HEKPLSFNPYKMLEELDKNSRAIKRRLERIKIPGVRAQGRYMDILCDAEV</entry></row></tbody></tgroup></table></tables><br /> (italic: nucleic acid editing domain)

0397Rhesus Macaque APOBEC-3H:

0398<tables id="TABLE-US-00070" num="00070"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 72)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MALLTAKTFSLQFNNKRRVNKPYYPRKALLCYQLTPQNGSTPTRGHLKNK</entry></row><row><entry></entry></row><row><entry>KKDHAEIRFINKIKSMGLDETQCYQVTCYLTWSPCPSCAGELVDFIKAHR</entry></row><row><entry></entry></row><row><entry>HLNLRIFASRLYYHWRPNYQEGLLLLCGSQVPVEVMGLPEFTDCWENFVD</entry></row><row><entry></entry></row><row><entry>HKEPPSFNPSEKLEELDKNSQAIKRRLERIKSRSVDVLENGLRSLQLGPV</entry></row><row><entry></entry></row><row><entry>TPSSSIRNSR</entry></row></tbody></tgroup></table></tables>

0399Human APOBEC-3D:

0400<tables id="TABLE-US-00071" num="00071"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 73)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLW</entry></row><row><entry></entry></row><row><entry>DTGVFRGPVLPKRQSNHRQEVYFRFEN<i>HAEMCFLSWFCGNRLPANRRFQI</i></entry></row><row><entry></entry></row><row><entry>TWFVSWNPCLPCVVKVTKFLAEHPNVTLTISAARLYYYRDRDWRWVLLRL</entry></row><row><entry></entry></row><row><entry>HKAGARVKIMDYEDFAYCWENFVCNEGQPFMPWYKFDDNYASLHRTLKEI</entry></row><row><entry></entry></row><row><entry>LRNPMEAMYPHIFYFHFKNLLKACGRNESWLCFTMEVTKHHSAVFRKRGV</entry></row><row><entry></entry></row><row><entry>FRNQVDPETHC<i>HAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPEC</i>AGEV</entry></row><row><entry></entry></row><row><entry>AEFLARHSNVNLTIFTARLCYFWDTDYQEGLCSLSQEGASVKIMGYKDFV</entry></row><row><entry></entry></row><row><entry>SCWKNFVYSDDEPFKPWKGLQTNFRLLKRRLREILQ</entry></row></tbody></tgroup></table></tables><br /> (italic: nucleic acid editing domain)

0401Human APOBEC-1:

0402<tables id="TABLE-US-00072" num="00072"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 74)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKI</entry></row><row><entry></entry></row><row><entry>WRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAI</entry></row><row><entry></entry></row><row><entry>REFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYY</entry></row><row><entry></entry></row><row><entry>HCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQ</entry></row><row><entry></entry></row><row><entry>NHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR</entry></row></tbody></tgroup></table></tables>

0403Mouse APOBEC-1:

0404<tables id="TABLE-US-00073" num="00073"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 75)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSV</entry></row><row><entry></entry></row><row><entry>WRHTSQNTSNHVEVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRHPYVTLFIYIARLYHHTDQRNRQGLRDLISSGVTIQIMTEQEYC</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYPPSNEAYWPRYPHLWVKLYVLELYCIILGLPPCLKILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTITLQTCHYQRIPPHLLWATGLK</entry></row></tbody></tgroup></table></tables>

0405Rat APOBEC-1:

0406<tables id="TABLE-US-00074" num="00074"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 76)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLK</entry></row></tbody></tgroup></table></tables>

0407Human APOBEC-2:

0408<tables id="TABLE-US-00075" num="00075"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 77)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MAQKEEAAVATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPAN</entry></row><row><entry></entry></row><row><entry>FFKFQFRNVEYSSGRNKTFLCYVVEAQGKGGQVQASRGYLEDEHAAAHAE</entry></row><row><entry></entry></row><row><entry>EAFFNTILPAFDPALRYNVTWYVSSSPCAACADRIIKTLSKTKNLRLLIL</entry></row><row><entry></entry></row><row><entry>VGRLFMWEEPEIQAALKKLKEAGCKLRIMKPQDFEYVWQNFVEQEEGESK</entry></row><row><entry></entry></row><row><entry>AFQPWEDIQENFLYYEEKLADILK</entry></row></tbody></tgroup></table></tables>

0409Mouse APOBEC-2:

0410<tables id="TABLE-US-00076" num="00076"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 78)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVN</entry></row><row><entry></entry></row><row><entry>FFKFQFRNVEYSSGRNKTFLCYVVEVQSKGGQAQATQGYLEDEHAGAHAE</entry></row><row><entry></entry></row><row><entry>EAFFNTILPAFDPALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLIL</entry></row><row><entry></entry></row><row><entry>VSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEYIWQNFVEQEEGESK</entry></row><row><entry></entry></row><row><entry>AFEPWEDIQENFLYYEEKLADILK</entry></row></tbody></tgroup></table></tables>

0411Rat APOBEC-2:

0412<tables id="TABLE-US-00077" num="00077"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 79)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVN</entry></row><row><entry></entry></row><row><entry>FFKFQFRNVEYSSGRNKTFLCYVVEAQSKGGQVQATQGYLEDEHAGAHAE</entry></row><row><entry></entry></row><row><entry>EAFFNTILPAFDPALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLIL</entry></row><row><entry></entry></row><row><entry>VSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEYLWQNFVEQEEGESK</entry></row><row><entry></entry></row><row><entry>AFEPWEDIQENFLYYEEKLADILK</entry></row></tbody></tgroup></table></tables>

0413Bovine APOBEC-2:

0414<tables id="TABLE-US-00078" num="00078"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 80)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPAH</entry></row><row><entry></entry></row><row><entry>YFKFQFRNVEYSSGRNKTFLCYVVEAQSKGGQVQASRGYLEDEHATNHAE</entry></row><row><entry></entry></row><row><entry>EAFFNSIMPTFDPALRYMVTWYVSSSPCAACADRIVKTLNKTKNLRLLIL</entry></row><row><entry></entry></row><row><entry>VGRLFMWEEPEIQAALRKLKEAGCRLRIMKPQDFEYIWQNFVEQEEGESK</entry></row><row><entry></entry></row><row><entry>AFEPWEDIQENFLYYEEKLADILK</entry></row></tbody></tgroup></table></tables>

0415<i>Petromyzon marinus </i>CDA1 (pmCDA1)

0416<tables id="TABLE-US-00079" num="00079"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 81)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFW</entry></row><row><entry></entry></row><row><entry>GYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADC</entry></row><row><entry></entry></row><row><entry>AEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNV</entry></row><row><entry></entry></row><row><entry>MVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKIL</entry></row><row><entry></entry></row><row><entry>HTTKSPAV</entry></row></tbody></tgroup></table></tables>

0417Human APOBEC3G D316R_D317R

0418<tables id="TABLE-US-00080" num="00080"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 82)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLD</entry></row><row><entry></entry></row><row><entry>AKIFRGQVYSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKC</entry></row><row><entry></entry></row><row><entry>TRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMK</entry></row><row><entry></entry></row><row><entry>IMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPP</entry></row><row><entry></entry></row><row><entry>TFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKH</entry></row><row><entry></entry></row><row><entry>GFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFIS</entry></row><row><entry></entry></row><row><entry>KNKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTF</entry></row><row><entry></entry></row><row><entry>VDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN</entry></row></tbody></tgroup></table></tables>

0419Human APOBEC3G chain A

0420<tables id="TABLE-US-00081" num="00081"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 83)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQA</entry></row><row><entry></entry></row><row><entry>PHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMA</entry></row><row><entry></entry></row><row><entry>KFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHC</entry></row><row><entry></entry></row><row><entry>WDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQ</entry></row></tbody></tgroup></table></tables>

0421Human APOBEC3G chain A D120R_D121R

0422<tables id="TABLE-US-00082" num="00082"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 84)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQA</entry></row><row><entry></entry></row><row><entry>PHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMA</entry></row><row><entry></entry></row><row><entry>KFISKNKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISIMTYSEFKHC</entry></row><row><entry></entry></row><row><entry>WDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQ</entry></row></tbody></tgroup></table></tables>

0423In some embodiments, fusion proteins as provided herein comprise the full-length amino acid of a nucleic acid editing enzyme, e.g., one of the sequences provided above. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length sequence of a nucleic acid editing enzyme, but only a fragment thereof. For example, in some embodiments, a fusion protein provided herein comprises a Cas9 domain and a fragment of a nucleic acid editing enzyme, e.g., wherein the fragment comprises a nucleic acid editing domain. Exemplary amino acid sequences of nucleic acid editing domains are shown in the sequences above as italicized letters, and additional suitable sequences of such domains will be apparent to those of skill in the art.

0424Additional suitable nucleic-acid editing enzyme sequences, e.g., deaminase enzyme and domain sequences, that can be used according to aspects of this invention, e.g., that can be fused to a nuclease-inactive Cas9 domain, will be apparent to those of skill in the art based on this disclosure. In some embodiments, such additional enzyme sequences include deaminase enzyme or deaminase domain sequences that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar to the sequences provided herein. Additional suitable Cas9 domains, variants, and sequences will also be apparent to those of skill in the art. Examples of such additional suitable Cas9 domains include, but are not limited to, D10A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains (see, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838 the entire contents of which are incorporated herein by reference). In some embodiments, the Cas9 comprises a histidine residue at position 840 of the amino acid sequence provided in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. The presence of the catalytic residue H840 restores the activity of the Cas9 to cleave the non-edited strand containing a G opposite the targeted C. Restoration of H840 does not result in the cleavage of the target strand containing the C.

0425Additional suitable strategies for generating fusion proteins comprising a Cas9 domain and a deaminase domain will be apparent to those of skill in the art based on this disclosure in combination with the general knowledge in the art. Suitable strategies for generating fusion proteins according to aspects of this disclosure using linkers or without the use of linkers will also be apparent to those of skill in the art in view of the instant disclosure and the knowledge in the art. For example, Gilbert et al., CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. <i>Cell. </i>2013; 154(2):442-51, showed that C-terminal fusions of Cas9 with VP64 using 2 NLS's as a linker (SPKKKRKVEAS, SEQ ID NO: 617), can be employed for transcriptional activation. Mali et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. <i>Nat Biotechnol. </i>2013; 31(9):833-8, reported that C-terminal fusions with VP64 without linker can be employed for transcriptional activation. And Maeder et al., CRISPR RNA-guided activation of endogenous human genes. <i>Nat Methods. </i>2013; 10: 977-979, reported that C-terminal fusions with VP64 using a Gly₄Ser (SEQ ID NO: 613) linker can be used as transcriptional activators. Recently, dCas9-FokI nuclease fusions have successfully been generated and exhibit improved enzymatic specificity as compared to the parental Cas9 enzyme (In Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. <i>Nat. Biotechnol. </i>2014; 32(6): 577-82, and in Tsai S Q, Wyvekens N, Khayter C, Foden J A, Thapar V, Reyon D, Goodwin M J, Aryee M J, Joung J K. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. <i>Nat Biotechnol. </i>2014; 32(6):569-76. PMID: 24770325 a SGSETPGTSESATPES (SEQ ID NO: 604) or a GGGGS (SEQ ID NO: 607) linker was used in FokI-dCas9 fusion proteins, respectively).

0426Some aspects of this disclosure provide fusion proteins comprising (i) a Cas9 enzyme or domain (e.g., a first protein); and (ii) a nucleic acid-editing enzyme or domain (e.g., a second protein). In some aspects, the fusion proteins provided herein further include (iii) a programmable DNA-binding protein, for example, a zinc-finger domain, a TALE, or a second Cas9 protein (e.g., a third protein). Without wishing to be bound by any particular theory, fusing a programmable DNA-binding protein (e.g., a second Cas9 protein) to a fusion protein comprising (i) a Cas9 enzyme or domain (e.g., a first protein); and (ii) a nucleic acid-editing enzyme or domain (e.g., a second protein) may be useful for improving specificity of the fusion protein to a target nucleic acid sequence, or for improving specificity or binding affinity of the fusion protein to bind target nucleic acid sequence that does not contain a canonical PAM (NGG) sequence. In some embodiments, the third protein is a Cas9 protein (e.g., a second Cas9 protein). In some embodiments, the third protein is any of the Cas9 proteins provided herein. In some embodiments, the third protein is fused to the fusion protein N-terminal to the Cas9 protein (e.g., the first protein). In some embodiments, the third protein is fused to the fusion protein C-terminal to the Cas9 protein (e.g., the first protein). In some embodiments, the Cas9 domain (e.g., the first protein) and the third protein (e.g., a second Cas9 protein) are fused via a linker (e.g., a second linker). In some embodiments, the linker comprises a (GGGGS)_n(SEQ ID NO: 607), a (G)_n(SEQ ID NO: 608), an (EAAAK)_n(SEQ ID NO: 609), a (GGS)_n(SEQ ID NO: 610), (SGGS)_n(SEQ ID NO: 606), a SGSETPGTSESATPES (SEQ ID NO: 604), a SGGS(GGS)_n(SEQ ID NO: 612), a SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605), or an (XP)_n(SEQ ID NO: 611) motif, or a combination of any of these, wherein n is independently an integer between 1 and 30. In some embodiments, the general architecture of exemplary napDNAbp fusion proteins provided herein comprises the structure: <ul id="ul0001" list-style="none"><li id="ul0001-0001" num="0427">[NH2]-[nucleic acid-editing enzyme or domain]-[napDNAbp]-[third protein]-[COOH];</li><li id="ul0001-0002" num="0428">[NH2]-[third protein]-[napDNAbp]-[nucleic acid-editing enzyme or domain]-[COOH];</li><li id="ul0001-0003" num="0429">[NH2]-[napDNAbp]-[nucleic acid-editing enzyme or domain]-[third protein]-[COOH];</li><li id="ul0001-0004" num="0430">[NH2]-[third protein]-[nucleic acid-editing enzyme or domain]-[napDNAbp]-[COOH];</li><li id="ul0001-0005" num="0431">[NH2]-[UGI]-[nucleic acid-editing enzyme or domain]-napDNAbp]-[third protein]-[COOH];</li><li id="ul0001-0006" num="0432">[NH2]-[UGI]-[third protein]-[napDNAbp]-[nucleic acid-editing enzyme or domain]-[COOH];</li><li id="ul0001-0007" num="0433">[NH2]-[UGI]-[napDNAbp]-[nucleic acid-editing enzyme or domain]-[third protein]-[COOH];</li><li id="ul0001-0008" num="0434">[NH2]-[UGI]-[third protein]-[nucleic acid-editing enzyme or domain]-[napDNAbp]-[COOH];</li><li id="ul0001-0009" num="0435">[NH2]-[nucleic acid-editing enzyme or domain]-[napDNAbp]-[third protein]-[UGI]-[COOH];</li><li id="ul0001-0010" num="0436">[NH2]-[third protein]-[napDNAbp]-[nucleic acid-editing enzyme or domain]-[UGI]-[COOH];</li><li id="ul0001-0011" num="0437">[NH2]-[NapDNAbp]-[nucleic acid-editing enzyme or domain]-[third protein]-[UGI]-[COOH];</li><li id="ul0001-0012" num="0438">[NH2]-[third protein]-[nucleic acid-editing enzyme or domain]-[NapDNAbp]-[UGI]-[COOH]; or</li><li id="ul0001-0013" num="0439">[NH2]-[nucleic acid-editing enzyme or domain]-[NapDNAbp]-[first UGI domain]-[second UGI domain]-[COOH]; <br /> wherein NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. In some embodiments, the “]-[” used in the general architecture above indicates the presence of an optional linker sequence. In other examples, the general architecture of exemplary NapDNAbp fusion proteins provided herein comprises the structure: </li><li id="ul0001-0014" num="0440">[NH2]-[nucleic acid-editing enzyme or domain]-[NapDNAbp]-[second NapDNAbp protein]-[COOH];</li><li id="ul0001-0015" num="0441">[NH2]-[second NapDNAbp protein]-[NapDNAbp]-[nucleic acid-editing enzyme or domain]-[COOH];</li><li id="ul0001-0016" num="0442">[NH2]-[NapDNAbp]-[nucleic acid-editing enzyme or domain]-[second NapDNAbp protein]-[COOH];</li><li id="ul0001-0017" num="0443">[NH2]-[second NapDNAbp protein]-[nucleic acid-editing enzyme or domain]-[NapDNAbp]-[COOH];</li><li id="ul0001-0018" num="0444">[NH2]-[UGI]-[nucleic acid-editing enzyme or domain]-[NapDNAbp]-[second NapDNAbp protein]-[COOH],</li><li id="ul0001-0019" num="0445">[NH2]-[UGI]-[second NapDNAbp protein]-[NapDNAbp]-[nucleic acid-editing enzyme or domain]-[COOH];</li><li id="ul0001-0020" num="0446">[NH2]-[UGI]-[NapDNAbp]-[nucleic acid-editing enzyme or domain]-[second NapDNAbp protein]-[COOH];</li><li id="ul0001-0021" num="0447">[NH2]-[UGI]-[second NapDNAbp protein]-[nucleic acid-editing enzyme or domain]-[NapDNAbp]-[COOH];</li><li id="ul0001-0022" num="0448">[NH2]-[nucleic acid-editing enzyme or domain]-[NapDNAbp]-[second NapDNAbp protein]-[UGI]-[COOH];</li><li id="ul0001-0023" num="0449">[NH2]-[second NapDNAbp protein]-[NapDNAbp]-[nucleic acid-editing enzyme or domain]-[UGI]-[COOH];</li><li id="ul0001-0024" num="0450">[NH2]-[NapDNAbp]-[nucleic acid-editing enzyme or domain]-[second NapDNAbp protein]-[UGI]-[COOH]; or</li><li id="ul0001-0025" num="0451">[NH2]-[second NapDNAbp protein]-[nucleic acid-editing enzyme or domain]-[NapDNAbp]-[UGI]-[COOH]; <br /> wherein NH₂is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. In some embodiments, the “]-[” used in the general architecture above indicates the presence of an optional linker sequence. In some embodiments, the second NapDNAbp is a dCas9 protein. In some examples, the general architecture of exemplary Cas9 fusion proteins provided herein comprises a structure as shown in <figref idref="DRAWINGS">FIG. 3</figref>. It should be appreciated that any of the proteins provided in any of the general architectures of exemplary Cas9 fusion proteins may be connected by one or more of the linkers provided herein. In some embodiments, the linkers are the same. In some embodiments, the linkers are different. In some embodiments, one or more of the proteins provided in any of the general architectures of exemplary Cas9 fusion proteins are not fused via a linker. In some embodiments, the fusion proteins further comprise a nuclear targeting sequence, for example a nuclear localization sequence. In some embodiments, fusion proteins provided herein further comprise a nuclear localization sequence (NLS). In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the third protein. In some embodiments, the NLS is fused to the C-terminus of the third protein. In some embodiments, the NLS is fused to the N-terminus of the Cas9 protein. In some embodiments, the NLS is fused to the C-terminus of the Cas9 protein. In some embodiments, the NLS is fused to the N-terminus of the nucleic acid-editing enzyme or domain. In some embodiments, the NLS is fused to the C-terminus of the nucleic acid-editing enzyme or domain. In some embodiments, the NLS is fused to the N-terminus of the UGI protein. In some embodiments, the NLS is fused to the C-terminus of the UGI protein. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker <br /> Uracil Glycosylase Inhibitor Fusion Proteins </li></ul>

0452Some aspects of the disclosure relate to fusion proteins that comprise a uracil glycosylase inhibitor (UGI) domain. In some embodiments, any of the fusion proteins provided herein that comprise a Cas9 domain (e.g., a nuclease active Cas9 domain, a nuclease inactive dCas9 domain, or a Cas9 nickase) may be further fused to a UGI domain either directly or via a linker. Some aspects of this disclosure provide deaminase-dCas9 fusion proteins, deaminase-nuclease active Cas9 fusion proteins and deaminase-Cas9 nickase fusion proteins with increased nucleobase editing efficiency. Without wishing to be bound by any particular theory, cellular DNA-repair response to the presence of U:G heteroduplex DNA may be responsible for the decrease in nucleobase editing efficiency in cells. For example, uracil DNA glycosylase (UDG) catalyzes removal of U from DNA in cells, which may initiate base excision repair, with reversion of the U:G pair to a C:G pair as the most common outcome. As demonstrated in the Examples below, Uracil DNA Glycosylase Inhibitor (UGI) may inhibit human UDG activity. Thus, this disclosure contemplates a fusion protein comprising dCas9-nucleic acid editing domain further fused to a UGI domain. This disclosure also contemplates a fusion protein comprising a Cas9 nickase-nucleic acid editing domain further fused to a UGI domain. It should be understood that the use of a UGI domain may increase the editing efficiency of a nucleic acid editing domain that is capable of catalyzing a C to U change. For example, fusion proteins comprising a UGI domain may be more efficient in deaminating C residues. In some embodiments, the fusion protein comprises the structure: <ul id="ul0002" list-style="none"><li id="ul0002-0001" num="0453">[deaminase]-[optional linker sequence]-[dCas9]-[optional linker sequence]-[UGI];</li><li id="ul0002-0002" num="0454">[deaminase]-[optional linker sequence]-[UGI]-[optional linker sequence]-[dCas9];</li><li id="ul0002-0003" num="0455">[UGI]-[optional linker sequence]-[deaminase]-[optional linker sequence]-[dCas9];</li><li id="ul0002-0004" num="0456">[UGI]-[optional linker sequence]-[dCas9]-[optional linker sequence]-[deaminase];</li><li id="ul0002-0005" num="0457">[dCas9]-[optional linker sequence]-[deaminase]-[optional linker sequence]-[UGI];</li><li id="ul0002-0006" num="0458">[dCas9]-[optional linker sequence]-[UGI]-[optional linker sequence]-[deaminase];</li><li id="ul0002-0007" num="0459">[deaminase]-[optional linker sequence]-[dCas9]-[optional linker sequence]-[first UGI]-[optional linker sequence]-[second UGI];</li><li id="ul0002-0008" num="0460">[deaminase]-[optional linker sequence]-[first UGI]-[optional linker segeunce]-[second UGI]-[optional linker sequence]-[dCas9];</li><li id="ul0002-0009" num="0461">[first UGI]-[optional linker sequence]-[second UGI]-[optional linker sequence]-[deaminase]-[optional linker sequence]-[dCas9];</li><li id="ul0002-0010" num="0462">[first UGI]-[optional linker sequence]-[second UGi]-[optional linker sequence]-[dCas9]-[optional linker sequence]-[deaminase];</li><li id="ul0002-0011" num="0463">[dCas9]-[optional linker sequence]-[deaminase]-[optional linker sequence]-[first UGI]-[optional linker sequence]-[second UGI]; or</li><li id="ul0002-0012" num="0464">[dCas9]-[optional linker sequence]-[first UGI]-[optional linker sequence]-[second UGI]-[optional linker sequence]-[deaminase].</li></ul>

0465In other embodiments, the fusion protein comprises the structure: <ul id="ul0003" list-style="none"><li id="ul0003-0001" num="0466">[deaminase]-[optional linker sequence]-[Cas9 nickase]-[optional linker sequence]-[UGI];</li><li id="ul0003-0002" num="0467">[deaminase]-[optional linker sequence]-[UGI]-[optional linker sequence]-[Cas9 nickase];</li><li id="ul0003-0003" num="0468">[UGI]-[optional linker sequence]-[deaminase]-[optional linker sequence]-[Cas9 nickase];</li><li id="ul0003-0004" num="0469">[UGI]-[optional linker sequence]-[Cas9 nickase]-[optional linker sequence]-[deaminase];</li><li id="ul0003-0005" num="0470">[Cas9 nickase]-[optional linker sequence]-[deaminase]-[optional linker sequence]-[UGI];</li><li id="ul0003-0006" num="0471">[Cas9 nickase]-[optional linker sequence]-[UGI]-[optional linker sequence]-[deaminase]</li><li id="ul0003-0007" num="0472">[deaminase]-[optional linker sequence]-[Cas9 nickase]-[optional linker sequence]-[first UGI]-[optional linker sequence]-[second UGI];</li><li id="ul0003-0008" num="0473">[deaminase]-[optional linker sequence]-[first UGI]-[optional linker sequence]-[second UGI]-[optional linker sequence]-[Cas9 nickase];</li><li id="ul0003-0009" num="0474">[first UGI]-[optional linker sequence]-[second UGI]-[optional linker sequence]-[deaminase]-[optional linker sequence]-[Cas9 nickase];</li><li id="ul0003-0010" num="0475">[first UGI]-[optional linker sequence]-[second UGi]-[optional linker sequence]-[Cas9 nickase]-[optional linker sequence]-[deaminase];</li><li id="ul0003-0011" num="0476">[Cas9 nickase]-[optional linker sequence]-[deaminase]-[optional linker sequence]-[first UGI]-[optional linker sequence]-[second UGI]; or</li><li id="ul0003-0012" num="0477">[Cas9 nickase]-[optional linker sequence]-[first UGI]-[optional linker sequence]-[second UGI]-[optional linker sequence]-[deaminase].</li></ul>

0478It should be appreciated that any of the fusion proteins described above may be comprised of (i) a nucleic acid programmable DNA binding protein (napDNAbp); (ii) a cytidine deaminase domain; and (iii) two or more UGI domains, wherein the two or more UGI domains may be adjacent (e.g., [first UGI]-[second UGI], wherein “-” is an optional linker) to one another in the construct, or the two or more UGI domains may be separated by the napDNAbp of (i) and/or the cytidine deaminase domain of (ii) (e.g., [first UGI]-[deaminase]-[second UGI], [first UGI]-[napDNAbp]-[second UGI], [first UGI]-[deaminase]-[napDNAbp]-[second UGI], ect., wherein “-” is an optional linker).

0479In another aspect, the fusion protein comprises: (i) a Cas9 enzyme or domain; (ii) a nucleic acid-editing enzyme or domain (e.g., a second protein) (e.g., a cytidine deaminase domain); (iii) a first uracil glycosylase inhibitor domain (UGI) (e.g., a third protein); and (iv) a second uracil glycosylase inhibitor domain (UGI) (e.g., a fourth protein). The first and second uracil glycosylase inhibitor domains (UGIs) may be the same or different. In some embodiments, the Cas9 domain (e.g., the first protein) and the deaminase (e.g., the second protein) are fused via a linker. In some embodiments, the Cas9 domain is fused to the C-terminus of the deaminase. In some embodiments, the Cas9 protein (e.g., the first protein) and the first UGI domain (e.g., the third protein) are fused via a linker (e.g., a second linker). In some embodiments, the first UGI domain is fused to the C-terminus of the Cas9 protein. In some embodiments, the first UGI domain (e.g., the third protein) and the second UGI domain (e.g., the forth protein) are fused via a linker (e.g., a third linker). In some embodiments, the second UGI domain is fused to the C-terminus of the first UGI domain. In some embodiments, the linker comprises a (GGGGS)_n(SEQ ID NO: 607), a (G)_n(SEQ ID NO: 608), an (EAAAK)_n(SEQ ID NO: 609), a (GGS)_n(SEQ ID NO: 610), (SGGS)_n(SEQ ID NO: 606), a SGSETPGTSESATPES (SEQ ID NO: 604), a SGGS(GGS)_n(SEQ ID NO: 612), a SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605), or an (XP)_n(SEQ ID NO: 611) motif, or a combination of any of these, wherein n is independently an integer between 1 and 30. In some embodiments, the first linker comprises an amino acid sequence of 1-50 amino acids. In some embodiments, the first linker comprises an amino acid sequence of 1-40 amino acids. In some embodiments, the first linker comprises an amino acid sequence of 1-35 amino acids. In some embodiments, the first linker comprises an amino acid sequence of 1-30 amino acids. In some embodiments, the first linker comprises an amino acid sequence of 1-20 amino acids. In some embodiments, the first linker comprises an amino acid sequence of 10-20 amino acids. In some embodiments, the first linker comprises an amino acid sequence of 30-40 amino acids. In some embodiments, the first linker comprises an amino acid sequence of 14, 16, or 18 amino acids. In some embodiments, the first linker comprises an amino acid sequence of 16 amino acids. In some embodiments, the first linker comprises an amino acid sequence of 30, 32, or 34 amino acids. In some embodiments, the first linker comprises an amino acid sequence of 32 amino acids. In some embodiments, the first linker comprises a SGSETPGTSESATPES (SEQ ID NO: 604) motif. In some embodiments, the first linker comprises a SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605) motif. In some embodiments, the second linker comprises an amino acid sequence of 1-50 amino acids. In some embodiments, the second linker comprises an amino acid sequence of 1-40 amino acids. In some embodiments, the second linker comprises an amino acid sequence of 1-35 amino acids. In some embodiments, the second linker comprises an amino acid sequence of 1-30 amino acids. In some embodiments, the second linker comprises an amino acid sequence of 1-20 amino acids. In some embodiments, the second linker comprises an amino acid sequence of 2-20 amino acids. In some embodiments, the second linker comprises an amino acid sequence of 2-10 amino acids. In some embodiments, the second linker comprises an amino acid sequence of 10-20 amino acids. In some embodiments, the second linker comprises an amino acid sequence of 2, 4, or 6 amino acids. In some embodiments, the second linker comprises an amino acid sequence of 7, 9, or 11 amino acids. In some embodiments, the second linker comprises an amino acid sequence of 14, 16, or 18 amino acids. In some embodiments, the second linker comprises an amino acid sequence of 4 amino acids. In some embodiments, the second linker comprises an amino acid sequence of 9 amino acids. In some embodiments, the second linker comprises an amino acid sequence of 16 amino acids. In some embodiments, the second linker comprises a (SGGS)_n(SEQ ID NO: 606) motif, wherein n is an integer between 1 and 30, inclusive. In some embodiments, the second linker comprises a (SGGS)_n(SEQ ID NO: 606) motif, wherein n is 1. In some embodiments, the second linker comprises a SGGS(GGS)_n(SEQ ID NO: 612) motif, wherein n is an integer between 1 and 30, inclusive. In some embodiments, the second linker comprises a SGGS(GGS)_n(SEQ ID NO: 612) motif, wherein n is 2. In some embodiments, the third linker comprises an amino acid sequence of 1-50 amino acids. In some embodiments, the third linker comprises an amino acid sequence of 1-40 amino acids. In some embodiments, the third linker comprises an amino acid sequence of 1-35 amino acids. In some embodiments, the third linker comprises an amino acid sequence of 1-30 amino acids. In some embodiments, the third linker comprises an amino acid sequence of 1-20 amino acids. In some embodiments, the third linker comprises an amino acid sequence of 2-20 amino acids. In some embodiments, the third linker comprises an amino acid sequence of 2-10 amino acids. In some embodiments, the third linker comprises an amino acid sequence of 10-20 amino acids. In some embodiments, the third linker comprises an amino acid sequence of 2, 4, or 6 amino acids. In some embodiments, the third linker comprises an amino acid sequence of 7, 9, or 11 amino acids. In some embodiments, the third linker comprises an amino acid sequence of 14, 16, or 18 amino acids. In some embodiments, the third linker comprises an amino acid sequence of 4 amino acids. In some embodiments, the third linker comprises an amino acid sequence of 9 amino acids. In some embodiments, the third linker comprises an amino acid sequence of 16 amino acids. In some embodiments, the third linker comprises a (SGGS)_n(SEQ ID NO: 606) motif, wherein n is an integer between 1 and 30, inclusive. In some embodiments, the third linker comprises a (SGGS)_n(SEQ ID NO: 606) motif, wherein n is 1. In some embodiments, the third linker comprises a SGGS(GGS)_n(SEQ ID NO: 612)motif, wherein n is an integer between 1 and 30, inclusive. In some embodiments, the third linker comprises a SGGS(GGS)_n(SEQ ID NO: 612) motif, wherein n is 2.

0480In some embodiments, the fusion protein comprises the structure: <ul id="ul0004" list-style="none"><li id="ul0004-0001" num="0481">[deaminase]-[optional linker sequence]-[dCas9]-[optional linker sequence]-[first UGI]-[optional linker sequence]-[second UGI];</li><li id="ul0004-0002" num="0482">[deaminase]-[optional linker sequence]-[Cas9 nickase]-[optional linker sequence]-[first UGI]-[optional linker sequence]-[second UGI]; or</li><li id="ul0004-0003" num="0483">[deaminase]-[optional linker sequence]-[Cas9]-[optional linker sequence]-[first UGI]-[optional linker sequence]-[second UGI].</li></ul>

0484In another aspect, the fusion protein comprises: (i) a Cas9 enzyme or domain; (ii) a nucleic acid-editing enzyme or domain (e.g., a second protein) (e.g., a cytidine deaminase domain); (iii) more than two uracil glycosylase inhibitor (UGI) domains.

0485In some embodiments, the fusion proteins provided herein do not comprise a linker sequence. In some embodiments, one or both of the optional linker sequences are present. In some embodiments, one, two, or three of the optional linker sequences are present.

0486In some embodiments, the “-” used in the general architecture above indicates the presence of an optional linker sequence. In some embodiments, the fusion proteins comprising a UGI further comprise a nuclear targeting sequence, for example a nuclear localization sequence. In some embodiments, fusion proteins provided herein further comprise a nuclear localization sequence (NLS). In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the UGI protein. In some embodiments, the NLS is fused to the C-terminus of the UGI protein. In some embodiments, the NLS is fused to the N-terminus of the Cas9 protein. In some embodiments, the NLS is fused to the C-terminus of the Cas9 protein. In some embodiments, the NLS is fused to the N-terminus of the deaminase. In some embodiments, the NLS is fused to the C-terminus of the deaminase. In some embodiments, the NLS is fused to the N-terminus of the second Cas9. In some embodiments, the NLS is fused to the C-terminus of the second Cas9. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises an amino acid sequence of any one of the NLS sequences provided or referenced herein. In some embodiments, the NLS comprises an amino acid sequence as set forth in SEQ ID NO: 614 or SEQ ID NO: 615.

0487In some embodiments, a UGI domain comprises a wild-type UGI or a UGI as set forth in SEQ ID NO: 134. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to a UGI or a UGI fragment. For example, in some embodiments, a UGI domain comprises a fragment of the amino acid sequence set forth in SEQ ID NO: 134. In some embodiments, a UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence as set forth in SEQ ID NO: 134. In some embodiments, a UGI comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 134 or an amino acid sequence homologous to a fragment of the amino acid sequence set forth in SEQ ID NO: 134. In some embodiments, proteins comprising UGI or fragments of UGI or homologs of UGI or UGI fragments are referred to as “UGI variants.” A UGI variant shares homology to UGI, or a fragment thereof. For example a UGI variant is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% identical to a wild type UGI or a UGI as set forth in SEQ ID NO: 134. In some embodiments, the UGI variant comprises a fragment of UGI, such that the fragment is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% to the corresponding fragment of wild-type UGI or a UGI as set forth in SEQ ID NO: 134. In some embodiments, the UGI comprises the following amino acid sequence:

0000>sp|P14739|UNGI_BPPB2 Uracil-DNA Glycosylase Inhibitor

0488<tables id="TABLE-US-00083" num="00083"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 134)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDES</entry></row><row><entry></entry></row><row><entry>TDENVMLLTSDAPEYKPWALVIQDSNGENKIKML</entry></row></tbody></tgroup></table></tables>

0489Suitable UGI protein and nucleotide sequences are provided herein and additional suitable UGI sequences are known to those in the art, and include, for example, those published in Wang et al., Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase. J. Biol. Chem. 264:1163-1171(1989); Lundquist et al., Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific carboxylic amino acids in complex formation with <i>Escherichia coli </i>uracil-DNA glycosylase. J. Biol. Chem. 272:21408-21419(1997); Ravishankar et al., X-ray analysis of a complex of <i>Escherichia coli </i>uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor. The structure elucidation of a prokaryotic UDG. Nucleic Acids Res. 26:4880-4887(1998); and Putnam et al., Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with <i>Escherichia coli </i>uracil-DNA glycosylase. J. Mol. Biol. 287:331-346(1999), the entire contents of each are incorporated herein by reference.

0490It should be appreciated that additional proteins may be uracil glycosylase inhibitors. For example, other proteins that are capable of inhibiting (e.g., sterically blocking) a uracil-DNA glycosylase base-excision repair enzyme are within the scope of this disclosure. Additionally, any proteins that block or inhibit base-excision repair as also within the scope of this disclosure. In some embodiments, the fusion proteins described herein comprise one UGI domain. In some embodiments, the fusion proteins described herein comprise more than one UGI domain. In some embodiments, the fusion proteins described herein comprise two UGI domains. In some embodiments, the fusion proteins described herein comprise more than two UGI domains. In some embodiments, a protein that binds DNA is used. In another embodiment, a substitute for UGI is used. In some embodiments, a uracil glycosylase inhibitor is a protein that binds single-stranded DNA. For example, a uracil glycosylase inhibitor may be a <i>Erwinia tasmaniensis </i>single-stranded binding protein. In some embodiments, the single-stranded binding protein comprises the amino acid sequence (SEQ ID NO: 135). In some embodiments, a uracil glycosylase inhibitor is a protein that binds uracil. In some embodiments, a uracil glycosylase inhibitor is a protein that binds uracil in DNA. In some embodiments, a uracil glycosylase inhibitor is a catalytically inactive uracil DNA-glycosylase protein. In some embodiments, a uracil glycosylase inhibitor is a catalytically inactive uracil DNA-glycosylase protein that does not excise uracil from the DNA. For example, a uracil glycosylase inhibitor is a UdgX. In some embodiments, the UdgX comprises the amino acid sequence (SEQ ID NO: 136). As another example, a uracil glycosylase inhibitor is a catalytically inactive UDG. In some embodiments, a catalytically inactive UDG comprises the amino acid sequence (SEQ ID NO: 137). It should be appreciated that other uracil glycosylase inhibitors would be apparent to the skilled artisan and are within the scope of this disclosure. In some embodiments, a uracil glycosylase inhibitor is a protein that is homologous to any one of SEQ ID NOs: 135-137 or 143-148. In some embodiments, a uracil glycosylase inhibitor is a protein that is at least 50% identical, at least 55% identical at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to any one of SEQ ID NOs: 135-137 or 143-148.

0000<i>Erwinia tasmaniensis </i>SSB (Themostable Single-Stranded DNA Binding Protein)

0491<tables id="TABLE-US-00084" num="00084"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 135)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKQTGETK</entry></row><row><entry></entry></row><row><entry>EKTEWHRVVLFGKLAEVAGEYLRKGSQVYIEGALQTRKWTDQAGVEKYTT</entry></row><row><entry></entry></row><row><entry>EVVVNVGGTMQMLGGRSQGGGASAGGQNGGSNNGWGQPQQPQGGNQFSGG</entry></row><row><entry></entry></row><row><entry>AQQQARPQQQPQQNNAPANNEPPIDFDDDIP</entry></row></tbody></tgroup></table></tables><br /> UdgX (Binds to Uracil in DNA but does not Excise)

0492<tables id="TABLE-US-00085" num="00085"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 136)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MAGAQDFVPHTADLAELAAAAGECRGCGLYRDATQAVFGAGGRSARIMMI</entry></row><row><entry></entry></row><row><entry>GEQPGDKEDLAGLPFVGPAGRLLDRALEAADIDRDALYVTNAVKHFKFTR</entry></row><row><entry></entry></row><row><entry>AAGGKRRIHKTPSRTEVVACRPWLIAEMTSVEPDVVVLLGATAAKALLGN</entry></row><row><entry></entry></row><row><entry>DFRVTQHRGEVLHVDDVPGDPALVATVHPSSLLRGPKEERESAFAGLVDD</entry></row><row><entry></entry></row><row><entry>LRVAADVRP</entry></row></tbody></tgroup></table></tables><br /> UDG (Catalytically Inactive Human UDG, Binds to Uracil in DNA but does not Excise)

0493<tables id="TABLE-US-00086" num="00086"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 137)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MIGQKTLYSFFSPSPARKRHAPSPEPAVQGTGVAGVPEESGDAAAIPAKK</entry></row><row><entry></entry></row><row><entry>APAGQEEPGTPPSSPLSAEQLDRIQRNKAAALLRLAARNVPVGFGESWKK</entry></row><row><entry></entry></row><row><entry>HLSGEFGKPYFIKLMGFVAEERKHYTVYPPPHQVFTWTQMCDIKDVKVVI</entry></row><row><entry></entry></row><row><entry>LGQEPYHGPNQAHGLCFSVQRPVPPPPSLENIYKELSTDIEDFVHPGHGD</entry></row><row><entry></entry></row><row><entry>LSGWAKQGVLLLNAVLTVRAHQANSHKERGWEQFTDAVVSWLNQNSNGLV</entry></row><row><entry></entry></row><row><entry>FLLWGSYAQKKGSAIDRKRHHVLQTAHPSPLSVYRGFFGCRHFSKTNELL</entry></row><row><entry></entry></row><row><entry>QKSGKKPIDWKEL</entry></row></tbody></tgroup></table></tables>

0494Additional single-stranded DNA binding proteins that can be used as a UGI are shown below. It should be appreciated that other single-stranded binding proteins may be used as a UGI, for example those described in Dickey T H, Altschuler S E, Wuttke D S. Single-stranded DNA-binding proteins:multiple domains for multiple functions. Structure. 2013 Jul. 2; 21(7):1074-84. <ul id="ul0005" list-style="none"><li id="ul0005-0001" num="0495">doi: 10.1016/j.str.2013.05.013. Review.; Marceau A H. Functions of single-strand DNA-binding proteins in DNA replication, recombination, and repair. Methods Mol Biol. 2012; 922:1-21. doi:</li><li id="ul0005-0002" num="0496">10.1007/978-1-62703-032-8_1.; Mijakovic, Ivan, et al.; Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine. Nucleic Acids Res 2006; 34 (5): 1588-1596. doi: 10.1093/nar/gkj514; Mumtsidu E, Makhov A M, Konarev P V, Svergun D I, Griffith J D, Tucker P A. Structural features of the single-stranded DNA-binding protein of Epstein-Barrvirus. J Struct Biol. 2008 February; 161(2):172-87. Epub 2007 Nov. 1; Nowak M, Olszewski M, Śpibida M, Kur J. Characterization of single-stranded DNA-binding proteins from the psychrophilic bacteria <i>Desulfotalea psychrophila, Flavobacterium psychrophilum, Psychrobacter arcticus, Psychrobacter cryohalolentis, Psychromonas ingrahamii, Psychroflexus torquis</i>, and</li><li id="ul0005-0003" num="0497"><i>Photobacterium profundum</i>. BMC Microbiol. 2014 Apr. 14; 14:91. doi: 10.1186/1471-2180-14-91; Tone T, Takeuchi A, Makino O. Single-stranded DNA binding protein Gp5 of <i>Bacillus subtilis </i>phage Φ29 is required for viral DNA replication in growth-temperature dependent fashion. Biosci Biotechnol Biochem. 2012; 76(12):2351-3. Epub 2012 Dec. 7; Wold. REPLICATION PROTEIN A: A Heterotrimeric, Single-Stranded DNA-Binding Protein Required for Eukaryotic DNA Metabolism. Annual Review of Biochem. 1997; 66:61-92. doi: 10.1146/annurev.biochem.66.1.61; Wu Y, Lu J, Kang T. Human single-stranded DNA binding proteins: guardians of genome stability. Acta Biochim Biophys Sin (Shanghai). 2016 July; 48(7):671-7. doi: 10.1093/abbs/gmw044. Epub 2016 May 23. Review; the entire contents of each are hereby incorporated by reference. <br /> mtSSB—SSBP1 Single Stranded DNA Binding Protein 1 [<i>Homo sapiens </i>(Human)](UniProtKB: Q04837; NP 001243439.1) </li></ul>

0498<tables id="TABLE-US-00087" num="00087"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 138)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MFRRPVLQVLRQFVRHESETTTSLVLERSLNRVHLLGRVGQDPVLRQVEG</entry></row><row><entry></entry></row><row><entry>KNPVTIFSLATNEMWRSGDSEVYQLGDVSQKTTWHRISVFRPGLRDVAYQ</entry></row><row><entry></entry></row><row><entry>YVKKGSRIYLEGKIDYGEYMDKNNVRRQATTIIADNIIFLSDQTKEKE</entry></row></tbody></tgroup></table></tables><br /> Single-Stranded DNA-Binding Protein 3 Isoform A [<i>Mus musculus</i>] (UniProtKB—Q9D032-1; NCBI Ref: NP_076161.2)

0499<tables id="TABLE-US-00088" num="00088"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 139)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MFAKGKGSAVPSDGQAREKLALYVYEYLLHVGAQKSAQTFLSEIRWEKNI</entry></row><row><entry></entry></row><row><entry>TLGEPPGFLHSWWCVFWDLYCAAPERRDTCEHSSEAKAFHDYSAAAAPSP</entry></row><row><entry></entry></row><row><entry>VLGNIPPNDGMPGGPIPPGFFQGPPGSQPSPHAQPPPHNPSSMMGPHSQP</entry></row><row><entry></entry></row><row><entry>FMSPRYAGGPRPPIRMGNQPPGGVPGTQPLLPNSMDPTRQQGHPNMGGSM</entry></row><row><entry></entry></row><row><entry>QRMNPPRGMGPMGPGPQNYGSGMRPPPNSLGPAMPGINMGPGAGRPWPNP</entry></row><row><entry></entry></row><row><entry>NSANSIPYSSSSPGTYVGPPGGGGPPGTPIMPSPADSTNSSDNIYTMINP</entry></row><row><entry></entry></row><row><entry>VPPGGSRSNFPMGPGSDGPMGGMGGMEPHHMNGSLGSGDIDGLPKNSPNN</entry></row><row><entry></entry></row><row><entry>ISGISNPPGTPRDDGELGGNFLHSFQNDNYSPSMTMSV</entry></row></tbody></tgroup></table></tables><br /> RPA 1—Replication Protein A 70 kDa DNA-Binding Subunit (UniProtKB: P27694; NCBI Ref: NM 002945.3)

0500<tables id="TABLE-US-00089" num="00089"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 140)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MVGQLSEGAIAAIMQKGDTNIKPILQVINIRPITTGNSPPRYRLLMSDGL</entry></row><row><entry></entry></row><row><entry>NTLSSFMLATQLNPLVEEEQLSSNCVCQIHRFIVNTLKDGRRVVILMELE</entry></row><row><entry></entry></row><row><entry>VLKSAEAVGVKIGNPVPYNEGLGQPQVAPPAPAASPAASSRPQPQNGSSG</entry></row><row><entry></entry></row><row><entry>MGSTVSKAYGASKTFGKAAGPSLSHTSGGTQSKVVPIASLTPYQSKWTIC</entry></row><row><entry></entry></row><row><entry>ARVTNKSQIRTWSNSRGEGKLFSLELVDESGEIRATAFNEQVDKFFPLIE</entry></row><row><entry></entry></row><row><entry>VNKVYYFSKGTLKIANKQFTAVKNDYEMTFNNETSVMPCEDDHHLPTVQF</entry></row><row><entry></entry></row><row><entry>DFTGIDDLENKSKDSLVDIIGICKSYEDATKITVRSNNREVAKRNIYLMD</entry></row><row><entry></entry></row><row><entry>TSGKVVTATLWGEDADKFDGSRQPVLAIKGARVSDFGGRSLSVLSSSTII</entry></row><row><entry></entry></row><row><entry>ANPDIPEAYKLRGWFDAEGQALDGVSISDLKSGGVGGSNTNWKTLYEVKS</entry></row><row><entry></entry></row><row><entry>ENLGQGDKPDYFSSVATVVYLRKENCMYQACPTQDCNKKVIDQQNGLYRC</entry></row><row><entry></entry></row><row><entry>EKCDTEFPNFKYRMILSVNIADFQENQWVTCFQESAEAILGQNAAYLGEL</entry></row><row><entry></entry></row><row><entry>KDKNEQAFEEVFQNANFRSFIFRVRVKVETYNDESRIKATVMDVKPVDYR</entry></row><row><entry></entry></row><row><entry>EYGRRLVMSIRRSALM</entry></row></tbody></tgroup></table></tables><br /> RPA 2—Replication Protein A 32 kDa Subunit (UniProtKB: P15927; NCBI Ref: NM_002946)

0501<tables id="TABLE-US-00090" num="00090"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 141)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MWNSGFESYGSSSYGGAGGYTQSPGGFGSPAPSQAEKKSRARAQHIVPCT</entry></row><row><entry></entry></row><row><entry>ISQLLSATLVDEVFRIGNVEISQVTIVGIIRHAEKAPTNIVYKIDDMTAA</entry></row><row><entry></entry></row><row><entry>PMDVRQWVDTDDTSSENTVVPPETYVKVAGHLRSFQNKKSLVAFKIMPLE</entry></row><row><entry></entry></row><row><entry>DMNEFTTHILEVINAHMVLSKANSQPSAGRAPISNPGMSEAGNFGGNSFM</entry></row><row><entry></entry></row><row><entry>PANGLTVAQNQVLNLIKACPRPEGLNFQDLKNQLKHMSVSSIKQAVDFLS</entry></row><row><entry></entry></row><row><entry>NEGHIYSTVDDDHFKSTDAE</entry></row></tbody></tgroup></table></tables><br /> RPA 3—Replication Protein A 14 kDa Subunit (UniProtKB: P35244; NCBI Ref: NM_002947.4)

0502<tables id="TABLE-US-00091" num="00091"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 142)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MVDMMDLPRSRINAGMLAQFIDKPVCFVGRLEKIHPTGKMFILSDGEGKN</entry></row><row><entry></entry></row><row><entry>GTIELMEPLDEEISGIVEVVGRVTAKATILCTSYVQFKEDSHPFDLGLYN</entry></row><row><entry></entry></row><row><entry>EAVKIIHDFPQFYPLGIVQHD</entry></row></tbody></tgroup></table></tables><br /> Bacterial Single-Stranded DNA-Binding Proteins: <br /> ssbA—Single-Stranded DNA-Binding Protein [<i>Bacillus subtilis </i>Subsp. <i>Subtilis </i>Str. 168](UniProtKB: P37455; NCBI Ref:)

0503<tables id="TABLE-US-00092" num="00092"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 143)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MLNRVVLVGRLTKDPELRYTPNGAAVATFTLAVNRTFTNQSGEREADFIN</entry></row><row><entry></entry></row><row><entry>CVTWRRQAENVANFLKKGSLAGVDGRLQTRNYENQQGQRVFVTEVQAESV</entry></row><row><entry></entry></row><row><entry>QFLEPKNGGGSGSGGYNEGNSGGGQYFGGGQNDNPFGGNQNNQRRNQGNS</entry></row><row><entry></entry></row><row><entry>FNDDPFANDGKPIDISDDDLPF</entry></row></tbody></tgroup></table></tables><br /> Single-Stranded DNA-Binding Protein 2 [<i>Streptomyces coelicolor </i>A3(2)] (UniProtKB: Q9X8U3; NCBI Ref: NP_628093.1)

0504<tables id="TABLE-US-00093" num="00093"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 144)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MAGETVITVVGNLVDDPELRFTPSGAAVAKFRVASTPRTFDRQTNEWKDG</entry></row><row><entry></entry></row><row><entry>ESLFLTCSVWRQAAENVAESLQRGMRVIVQGRLKQRSYEDREGVKRTVYE</entry></row><row><entry></entry></row><row><entry>LDVDEVGASLRSATAKVTKTSGQGRGGQGGYGGGGGGQGGGGWGGGPGGG</entry></row><row><entry></entry></row><row><entry>QQGGGAPADDPWATGGAPAGGQQGGGGQGGGGWGGGSGGGGGYSDEPPF</entry></row></tbody></tgroup></table></tables><br /> Single-Stranded DNA-Binding Protein [<i>Streptococcus pneumoniae </i>R6] (UniProtKB: P66855; NCBI Ref: NP_358988.1)

0505<tables id="TABLE-US-00094" num="00094"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 145)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MINNVVLVGRMTRDAELRYTPSNVAVATFTLAVNRTFKSQNGEREADFIN</entry></row><row><entry></entry></row><row><entry>VVMWRQQAENLANWAKKGSLIGVTGRIQTRSYDNQQGQRVYVTEVVAENF</entry></row><row><entry></entry></row><row><entry>QMLESRSVREGHTGGAYSAPTANYSAPTNSVPDFSRNENPFGATNPLDIS</entry></row><row><entry></entry></row><row><entry>DDDLPF</entry></row></tbody></tgroup></table></tables><br /> Viral Single-Stranded DNA-Binding Proteins: <br /> Single-Stranded DNA-Binding Protein [Human Alphaherpesvirus 1] (UniProtKB: P04296; NCBI Ref: YP_009137104.1)

0506<tables id="TABLE-US-00095" num="00095"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 146)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>METKPKTATTIKVPPGPLGYVYARACPSEGIELLALLSARSGDSDVAVAP</entry></row><row><entry></entry></row><row><entry>LVVGLTVESGFEANVAVVVGSRTTGLGGTAVSLKLTPSHYSSSVYVFHGG</entry></row><row><entry></entry></row><row><entry>RHLDPSTQAPNLTRLCERARRHFGFSDYTPRPGDLKHETTGEALCERLGL</entry></row><row><entry></entry></row><row><entry>DPDRALLYLVVTEGFKEAVCINNTFLHLGGSDKVTIGGAEVHRIPVYPLQ</entry></row><row><entry></entry></row><row><entry>LFMPDFSRVIAEPFNANHRSIGENFTYPLPFFNRPLNRLLFEAVVGPAAV</entry></row><row><entry></entry></row><row><entry>ALRCRNVDAVARAAAHLAFDENHEGAALPADITFTAFEASQGKTPRGGRD</entry></row><row><entry></entry></row><row><entry>GGGKGPAGGFEQRLASVMAGDAALALESIVSMAVFDEPPTDISAWPLFEG</entry></row><row><entry></entry></row><row><entry>QDTAAARANAVGAYLARAAGLVGAMVFSTNSALHLTEVDDAGPADPKDHS</entry></row><row><entry></entry></row><row><entry>KPSFYRFFLVPGTHVAANPQVDREGHVVPGFEGRPTAPLVGGTQEFAGEH</entry></row><row><entry></entry></row><row><entry>LAMLCGFSPALLAKMLFYLERCDGGVIVGRQEMDVFRYVADSNQTDVPCN</entry></row><row><entry></entry></row><row><entry>LCTFDTRHACVHTTLMRLRARHPKFASAARGAIGVFGTMNSMYSDCDVLG</entry></row><row><entry></entry></row><row><entry>NYAAFSALKRADGSETARTIMQETYRAATERVMAELETLQYVDQAVPTAM</entry></row><row><entry></entry></row><row><entry>GRLETIITNREALHTVVNNVRQVVDREVEQLMRNLVEGRNFKFRDGLGEA</entry></row><row><entry></entry></row><row><entry>NHAMSLTLDPYACGPCPLLQLLGRRSNLAVYQDLALSQCHGVFAGQSVEG</entry></row><row><entry></entry></row><row><entry>RNFRNQFQPVLRRRVMDMFNNGFLSAKTLTVALSEGAAICAPSLTAGQTA</entry></row><row><entry></entry></row><row><entry>PAESSFEGDVARVTLGFPKELRVKSRVLFAGASANASEAAKARVASLQSA</entry></row><row><entry></entry></row><row><entry>YQKPDKRVDILLGPLGFLLKQFHAAIFPNGKPPGSNQPNPQWFWTALQRN</entry></row><row><entry></entry></row><row><entry>QLPARLLSREDIETIAFIKKFSLDYGAINFINLAPNNVSELAMYYMANQI</entry></row><row><entry></entry></row><row><entry>LRYCDHSTYFINTLTAIIAGSRRPPSVQAAAAWSAQGGAGLEAGARALMD</entry></row><row><entry></entry></row><row><entry>AVDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQA</entry></row><row><entry></entry></row><row><entry>GNWASLMGGKNACPLLIFDRTRKFVLACPRAGFVCAASSLGGGAHESSLC</entry></row><row><entry></entry></row><row><entry>EQLRGIISEGGAAVASSVFVATVKSLGPRTQQLQIEDWLALLEDEYLSEE</entry></row><row><entry></entry></row><row><entry>MMELTARALERGNGEWSTDAALEVAHEAEALVSQLGNAGEVFNFGDFGCE</entry></row><row><entry></entry></row><row><entry>DDNATPFGGPGAPGPAFAGRKRAFHGDDPFGEGPPDKKGDLTLDML</entry></row></tbody></tgroup></table></tables><br /> Single-Stranded DNA-Binding Protein from <i>Bacillus </i>Virus Phi29 (UniProtKB: Q38504.1; NCBI Ref: YP_002004532.1)

0507<tables id="TABLE-US-00096" num="00096"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 147)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MENTNIVKATFDTETLEGQIKIFNAQTGGGQSFKNLPDGTIIEANAIAQY</entry></row><row><entry></entry></row><row><entry>KQVSDTYGDAKEETVTTIFAADGSLYSAISKTVAEAASDLIDLVTRHKLE</entry></row><row><entry></entry></row><row><entry>TFKVKVVQGTSSKGNVFFSLQLSL</entry></row></tbody></tgroup></table></tables><br /> Single Stranded DNA Binding Protein [<i>Burkholderia </i>Virus DC1] (UniProtKB: I6NRL7; NCBI Ref: YP_006589943.1)

0508<tables id="TABLE-US-00097" num="00097"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 148)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MASVNKVILVGNLGADPETRYLPSGDAISNIRLATTDRYKDKASGEMKES</entry></row><row><entry></entry></row><row><entry>TEWHRVSFFGRLAEIVDEYLRKGAPVYIEGRIRTRKWQDNAGQDRYTTEI</entry></row><row><entry></entry></row><row><entry>VAEKMQMLGDRRDGGERQQRAPQQQQQRTQRNGYADATGRAQPSQRPAAG</entry></row><row><entry></entry></row><row><entry>GGFDEMDDDIPF</entry></row></tbody></tgroup></table></tables>

0509In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytosine deaminase or a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase. In some embodiments, the deaminase is an APOBEC2 deaminase. In some embodiments, the deaminase is an APOBEC3 deaminase. In some embodiments, the deaminase is an APOBEC3A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase. In some embodiments, the deaminase is an APOBEC3E deaminase. In some embodiments, the deaminase is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deminase is a rat APOBEC1 (SEQ ID NO: 74). In some embodiments, the deminase is a human APOBEC1 (SEQ ID No: 76). In some embodiments, the deaminase is a <i>Petromyzon marinus </i>cytidine deaminase 1 (pmCDA1). In some embodiments, the deminase is a human APOBEC3G (SEQ ID NO: 60). In some embodiments, the deaminase is a fragment of the human APOBEC3G (SEQ ID NO: 83). In some embodiments, the deaminase is a human APOBEC3G variant comprising a D316R_D317R mutation (SEQ ID NO: 82). In some embodiments, the deaminase is a fragment of the human APOBEC3G and comprising mutations corresponding to the D316R_D317R mutations in SEQ ID NO: 60 (SEQ ID NO: 84).

0510In some embodiments, the linker comprises a (GGGS)_n(SEQ ID NO: 613), (GGGGS)_n(SEQ ID NO: 607), a (G)_n(SEQ ID NO: 608), an (EAAAK)_n(SEQ ID NO: 609), a (GGS)_n(SEQ ID NO: 610), an SGSETPGTSESATPES (SEQ ID NO: 604), or an (XP)_n(SEQ ID NO: 611) motif, or a combination of any of these, wherein n is independently an integer between 1 and 30.

0511Suitable UGI protein and nucleotide sequences are provided herein and additional suitable UGI sequences are known to those in the art, and include, for example, those published in Wang et al., Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase. <i>J. Biol. Chem. </i>264:1163-1171(1989); Lundquist et al., Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific carboxylic amino acids in complex formation with <i>Escherichia coli </i>uracil-DNA glycosylase. <i>J. Biol. Chem. </i>272:21408-21419(1997); Ravishankar et al., X-ray analysis of a complex of <i>Escherichia coli </i>uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor. The structure elucidation of a prokaryotic UDG. <i>Nucleic Acids Res. </i>26:4880-4887(1998); and Putnam et al., Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with <i>Escherichia coli </i>uracil-DNA glycosylase. <i>J. Mol. Biol. </i>287:331-346(1999), the entire contents of which are incorporated herein by reference. In some embodiments, the optional linker comprises a (GGS)_n(SEQ ID NO: 610) motif, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the optional linker comprises a (GGS)_n(SEQ ID NO: 610) motif, wherein n is 1, 3, or 7. In some embodiments, the optional linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604), which is also referred to as the XTEN linker in the Examples.

0512In some embodiments, a Cas9 nickase may further facilitate the removal of a base on the non-edited strand in an organism whose genome is edited in vivo. The Cas9 nickase, as described herein, may comprise a D10A mutation in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. In some embodiments, the Cas9 nickase of this disclosure may comprise a histidine at mutation 840 of SEQ ID NO: 6, or a corresponding residue in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein. Such fusion proteins comprising the Cas9 nickase, can cleave a single strand of the target DNA sequence, e.g., the strand that is not being edited. Without wishing to be bound by any particular theory, this cleavage may inhibit mis-match repair mechanisms that reverse a C to U edit made by the deaminase.

0000Cas9 Complexes with Guide RNAs

0513Some aspects of this disclosure provide complexes comprising any of the fusion proteins provided herein, and a guide RNA bound to a Cas9 domain (e.g., a dCas9, a nuclease active Cas9, or a Cas9 nickase) of fusion protein.

0514In some embodiments, the guide RNA is from 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some embodiments, the guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is a sequence in the genome of a mammal. In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3′ end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the guide RNA is complementary to a sequence associated with a disease or disorder.

0000Methods of Using Cas9 Fusion Proteins

0515Some aspects of this disclosure provide methods of using the Cas9 proteins, fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule (a) with any of the Cas9 proteins or fusion proteins provided herein, and with at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence; or (b) with a Cas9 protein, a Cas9 fusion protein, or a Cas9 protein or fusion protein complex with at least one gRNA as provided herein. In some embodiments, the 3′ end of the target sequence is not immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence.

0516In some embodiments, the target DNA sequence comprises a sequence associated with a disease or disorder. In some embodiments, the target DNA sequence comprises a point mutation associated with a disease or disorder. In some embodiments, the activity of the Cas9 protein, the Cas9 fusion protein, or the complex results in a correction of the point mutation. In some embodiments, the target DNA sequence comprises a T→C point mutation associated with a disease or disorder, and wherein the deamination of the mutant C base results in a sequence that is not associated with a disease or disorder. In some embodiments, the target DNA sequence encodes a protein and wherein the point mutation is in a codon and results in a change in the amino acid encoded by the mutant codon as compared to the wild-type codon. In some embodiments, the deamination of the mutant C results in a change of the amino acid encoded by the mutant codon. In some embodiments, the deamination of the mutant C results in the codon encoding the wild-type amino acid. In some embodiments, the contacting is in vivo in a subject. In some embodiments, the subject has or has been diagnosed with a disease or disorder. In some embodiments, the disease or disorder is cystic fibrosis, phenylketonuria, epidermolytic hyperkeratosis (EHK), Charcot-Marie-Toot disease type 4J, neuroblastoma (NB), von Willebrand disease (vWD), myotonia congenital, hereditary renal amyloidosis, dilated cardiomyopathy (DCM), hereditary lymphedema, familial Alzheimer's disease, HIV, Prion disease, chronic infantile neurologic cutaneous articular syndrome (CINCA), desmin-related myopathy (DRM), a neoplastic disease associated with a mutant PI3KCA protein, a mutant CTNNB1 protein, a mutant HRAS protein, or a mutant p53 protein.

0517Some embodiments provide methods for using the Cas9 DNA editing fusion proteins provided herein. In some embodiments, the fusion protein is used to introduce a point mutation into a nucleic acid by deaminating a target nucleobase, e.g., a C residue. In some embodiments, the deamination of the target nucleobase results in the correction of a genetic defect, e.g., in the correction of a point mutation that leads to a loss of function in a gene product. In some embodiments, the genetic defect is associated with a disease or disorder, e.g., a lysosomal storage disorder or a metabolic disease, such as, for example, type I diabetes. In some embodiments, the methods provided herein are used to introduce a deactivating point mutation into a gene or allele that encodes a gene product that is associated with a disease or disorder. For example, in some embodiments, methods are provided herein that employ a Cas9 DNA editing fusion protein to introduce a deactivating point mutation into an oncogene (e.g., in the treatment of a proliferative disease). A deactivating mutation may, in some embodiments, generate a premature stop codon in a coding sequence, which results in the expression of a truncated gene product, e.g., a truncated protein lacking the function of the full-length protein.

0518In some embodiments, the purpose of the methods provide herein is to restore the function of a dysfunctional gene via genome editing. The Cas9 deaminase fusion proteins provided herein can be validated for gene editing-based human therapeutics in vitro, e.g., by correcting a disease-associated mutation in human cell culture. It will be understood by the skilled artisan that the fusion proteins provided herein, e.g., the fusion proteins comprising a Cas9 domain and a nucleic acid deaminase domain can be used to correct any single point T→C or A→G mutation. In the first case, deamination of the mutant C back to U corrects the mutation, and in the latter case, deamination of the C that is base-paired with the mutant G, followed by a round of replication, corrects the mutation.

0519An exemplary disease-relevant mutation that can be corrected by the provided fusion proteins in vitro or in vivo is the H1047R (A3140G) polymorphism in the PI3KCA protein. The phosphoinositide-3-kinase, catalytic alpha subunit (PI3KCA) protein acts to phosphorylate the 3-OH group of the inositol ring of phosphatidylinositol. The PI3KCA gene has been found to be mutated in many different carcinomas, and thus it is considered to be a potent oncogene.³⁷In fact, the A3140G mutation is present in several NCI-60 cancer cell lines, such as, for example, the HCT116, SKOV3, and T47D cell lines, which are readily available from the American Type Culture Collection (ATCC).³⁸

0520In some embodiments, a cell carrying a mutation to be corrected, e.g., a cell carrying a point mutation, e.g., an A3140G point mutation in exon 20 of the PI3KCA gene, resulting in a H1047R substitution in the PI3KCA protein, is contacted with an expression construct encoding a Cas9 deaminase fusion protein and an appropriately designed sgRNA targeting the fusion protein to the respective mutation site in the encoding PI3KCA gene. Control experiments can be performed where the sgRNAs are designed to target the fusion enzymes to non-C residues that are within the PI3KCA gene. Genomic DNA of the treated cells can be extracted, and the relevant sequence of the PI3KCA genes PCR amplified and sequenced to assess the activities of the fusion proteins in human cell culture.

0521It will be understood that the example of correcting point mutations in PI3KCA is provided for illustration purposes and is not meant to limit the instant disclosure. The skilled artisan will understand that the instantly disclosed DNA-editing fusion proteins can be used to correct other point mutations and mutations associated with other cancers and with diseases other than cancer including other proliferative diseases.

0522The successful correction of point mutations in disease-associated genes and alleles opens up new strategies for gene correction with applications in therapeutics and basic research. Site-specific single-base modification systems like the disclosed fusions of Cas9 and deaminase enzymes or domains also have applications in “reverse” gene therapy, where certain gene functions are purposely suppressed or abolished. In these cases, site-specifically mutating Trp (TGG), Gln (CAA and CAG), or Arg (CGA) residues to premature stop codons (TAA, TAG, TGA) can be used to abolish protein function in vitro, ex vivo, or in vivo.

0523The instant disclosure provides methods for the treatment of a subject diagnosed with a disease associated with or caused by a point mutation that can be corrected by a Cas9 DNA editing fusion protein provided herein. For example, in some embodiments, a method is provided that comprises administering to a subject having such a disease, e.g., a cancer associated with a PI3KCA point mutation as described above, an effective amount of a Cas9 deaminase fusion protein that corrects the point mutation or introduces a deactivating mutation into the disease-associated gene. In some embodiments, the disease is a proliferative disease. In some embodiments, the disease is a genetic disease. In some embodiments, the disease is a neoplastic disease. In some embodiments, the disease is a metabolic disease. In some embodiments, the disease is a lysosomal storage disease. Other diseases that can be treated by correcting a point mutation or introducing a deactivating mutation into a disease-associated gene will be known to those of skill in the art, and the disclosure is not limited in this respect.

0524The instant disclosure provides methods for the treatment of additional diseases or disorders, e.g., diseases or disorders that are associated or caused by a point mutation that can be corrected by deaminase-mediated gene editing. Some such diseases are described herein, and additional suitable diseases that can be treated with the strategies and fusion proteins provided herein will be apparent to those of skill in the art based on the instant disclosure. Exemplary suitable diseases and disorders are listed below. It will be understood that the numbering of the specific positions or residues in the respective sequences depends on the particular protein and numbering scheme used. Numbering might be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. Exemplary suitable diseases and disorders include, without limitation, cystic fibrosis (see, e.g., Schwank et al., Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. <i>Cell stem cell. </i>2013; 13: 653-658; and Wu et. al., Correction of a genetic disease in mouse via use of CRISPR-Cas9<i>. Cell stem cell. </i>2013; 13: 659-662, neither of which uses a deaminase fusion protein to correct the genetic defect); phenylketonuria—e.g., phenylalanine to serine mutation at position 835 (mouse) or 240 (human) or a homologous residue in phenylalanine hydroxylase gene (T>C mutation)—see, e.g., McDonald et al., <i>Genomics. </i>1997; 39:402-405; Bernard-Soulier syndrome (BSS)—e.g., phenylalanine to serine mutation at position 55 or a homologous residue, or cysteine to arginine at residue 24 or a homologous residue in the platelet membrane glycoprotein IX (T>C mutation)—see, e.g., Noris et al., <i>British Journal of Haematology. </i>1997; 97: 312-320, and Ali et al., <i>Hematol. </i>2014; 93: 381-384; epidermolytic hyperkeratosis (EHK)—e.g., leucine to proline mutation at position 160 or 161 (if counting the initiator methionine) or a homologous residue in keratin 1 (T>C mutation)—see, e.g., Chipev et al., <i>Cell. </i>1992; 70: 821-828, see also accession number P04264 in the UNIPROT database at www[dot]uniprot[dot]org; chronic obstructive pulmonary disease (COPD)—e.g., leucine to proline mutation at position 54 or 55 (if counting the initiator methionine) or a homologous residue in the processed form of α₁-antitrypsin or residue 78 in the unprocessed form or a homologous residue (T>C mutation) —see, e.g., Poller et al., <i>Genomics. </i>1993; 17: 740-743, see also accession number P01011 in the UNIPROT database; Charcot-Marie-Toot disease type 4J—e.g., isoleucine to threonine mutation at position 41 or a homologous residue in <figref idref="DRAWINGS">FIG. 4</figref> (T>C mutation)—see, e.g., Lenk et al., PLoS Genetics. 2011; 7: e1002104; neuroblastoma (NB)—e.g., leucine to proline mutation at position 197 or a homologous residue in Caspase-9 (T>C mutation)—see, e.g., Kundu et al., 3 <i>Biotech. </i>2013, 3:225-234; von Willebrand disease (vWD)—e.g., cysteine to arginine mutation at position 509 or a homologous residue in the processed form of von Willebrand factor, or at position 1272 or a homologous residue in the unprocessed form of von Willebrand factor (T>C mutation)—see, e.g., Lavergne et al., <i>Br. J. Haematol. </i>1992, see also accession number P04275 in the UNIPROT database; 82: 66-72; myotonia congenital—e.g., cysteine to arginine mutation at position 277 or a homologous residue in the muscle chloride channel gene CLCN1 (T>C mutation)—see, e.g., Weinberger et al., <i>The J. of Physiology. </i>2012; 590: 3449-3464; hereditary renal amyloidosis—e.g., stop codon to arginine mutation at position 78 or a homologous residue in the processed form of apolipoprotein AII or at position 101 or a homologous residue in the unprocessed form (T>C mutation)—see, e.g., Yazaki et al., <i>Kidney Int. </i>2003; 64: 11-16; dilated cardiomyopathy (DCM)—e.g., tryptophan to Arginine mutation at position 148 or a homologous residue in the FOXD4 gene (T>C mutation), see, e.g., Minoretti et. al., <i>Int. J. of Mol. Med. </i>2007; 19: 369-372; hereditary lymphedema—e.g., histidine to arginine mutation at position 1035 or a homologous residue in VEGFR3 tyrosine kinase (A>G mutation), see, e.g., Irrthum et al., <i>Am. J. Hum. Genet. </i>2000; 67: 295-301; familial Alzheimer's disease—e.g., isoleucine to valine mutation at position 143 or a homologous residue in presenilin1 (A>G mutation), see, e.g., Gallo et. al., <i>J. Alzheimer's disease. </i>2011; 25: 425-431; Prion disease—e.g., methionine to valine mutation at position 129 or a homologous residue in prion protein (A>G mutation)—see, e.g., Lewis et. al., <i>J. of General Virology. </i>2006; 87: 2443-2449; chronic infantile neurologic cutaneous articular syndrome (CINCA)—e.g., Tyrosine to Cysteine mutation at position 570 or a homologous residue in cryopyrin (A>G mutation)—see, e.g., Fujisawa et. al. <i>Blood. </i>2007; 109: 2903-2911; and desmin-related myopathy (DRM)—e.g., arginine to glycine mutation at position 120 or a homologous residue in αβ crystallin (A>G mutation)—see, e.g., Kumar et al., <i>J. Biol. Chem. </i>1999; 274: 24137-24141. The entire contents of all references and database entries is incorporated herein by reference.

0525It will be apparent to those of skill in the art that in order to target a Cas9:nucleic acid editing enzyme/domain fusion protein as disclosed herein to a target site, e.g., a site comprising a point mutation to be edited, it is typically necessary to co-express the Cas9:nucleic acid editing enzyme/domain fusion protein together with a guide RNA, e.g., an sgRNA. As explained in more detail elsewhere herein, a guide RNA typically comprises a tracrRNA framework allowing for Cas9 binding, and a guide sequence, which confers sequence specificity to the Cas9:nucleic acid editing enzyme/domain fusion protein. In some embodiments, the guide RNA comprises a structure 5′-[guide sequence]-guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu u-3′ (SEQ ID NO: 618), wherein the guide sequence comprises a sequence that is complementary to the target sequence. The guide sequence is typically 20 nucleotides long. The sequences of suitable guide RNAs for targeting Cas9:nucleic acid editing enzyme/domain fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting Cas9:nucleic acid editing enzyme/domain fusion proteins to specific target sequences are provided below.

0000Base Editor Efficiency

0526Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of modifying a specific nucleotide base without generating a significant proportion of indels. An “indel”, as used herein, refers to the insertion or deletion of a nucleotide base within a nucleic acid. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. In some embodiments, it is desirable to generate base editors that efficiently modify (e.g. mutate or deaminate) a specific nucleotide within a nucleic acid, without generating a large number of insertions or deletions (i.e., indels) in the nucleic acid. In certain embodiments, any of the base editors provided herein are capable of generating a greater proportion of intended modifications (e.g., point mutations or deaminations) versus indels. In some embodiments, the base editors provided herein are capable of generating a ratio of intended point mutations to indels that is greater than 1:1. In some embodiments, the base editors provided herein are capable of generating a ratio of intended point mutations to indels that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1, or more. The number of intended mutations and indels may be determined using any suitable method, for example the methods used in the below Examples.

0527In some embodiments, the base editors provided herein are capable of limiting formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor. In some embodiments, any of the base editors provided herein are capable of limiting the formation of indels at a region of a nucleic acid to less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, or less than 20%. The number of indels formed at a nucleic acid region may depend on the amount of time a nucleic acid (e.g., a nucleic acid within the genome of a cell) is exposed to a base editor. In some embodiments, an number or proportion of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a nucleic acid (e.g., a nucleic acid within the genome of a cell) to a base editor.

0528Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of efficiently generating an intended mutation, such as a point mutation, in a nucleic acid (e.g. a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations. In some embodiments, a intended mutation is a mutation that is generated by a specific base editor bound to a gRNA, specifically designed to generate the intended mutation. In some embodiments, the intended mutation is a mutation associated with a disease or disorder. In some embodiments, the intended mutation is a cytosine (C) to thymine (T) point mutation associated with a disease or disorder. In some embodiments, the intended mutation is a guanine (G) to adenine (A) point mutation associated with a disease or disorder. In some embodiments, the intended mutation is a cytosine (C) to thymine (T) point mutation within the coding region of a gene. In some embodiments, the intended mutation is a guanine (G) to adenine (A) point mutation within the coding region of a gene. In some embodiments, the intended mutation is a point mutation that generates a stop codon, for example, a premature stop codon within the coding region of a gene. In some embodiments, the intended mutation is a mutation that eliminates a stop codon. In some embodiments, the intended mutation is a mutation that alters the splicing of a gene. In some embodiments, the intended mutation is a mutation that alters the regulatory sequence of a gene (e.g., a gene promotor or gene repressor). In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point mutations:unintended point mutations) that is greater than 1:1. In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point mutations:unintended point mutations) that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least 500:1, or at least 1000:1, or more. It should be appreciated that the characteristics of the base editors described in the “Base Editor Efficiency” section, herein, may be applied to any of the fusion proteins, or methods of using the fusion proteins provided herein.

0000Methods for Editing Nucleic Acids

0529Some aspects of the disclosure provide methods for editing a nucleic acid. In some embodiments, the method is a method for editing a nucleobase of a nucleic acid (e.g., a base pair of a double-stranded DNA sequence). In some embodiments, the method comprises the steps of: a) contacting a target region of a nucleic acid (e.g., a double-stranded DNA sequence) with a complex comprising a base editor (e.g., a Cas9 domain fused to a cytidine deaminase domain) and a guide nucleic acid (e.g., gRNA), wherein the target region comprises a targeted nucleobase pair, b) inducing strand separation of said target region, c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase, and d) cutting no more than one strand of said target region, where a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase; and the method results in less than 20% indel formation in the nucleic acid. It should be appreciated that in some embodiments, step b is omitted. In some embodiments, the first nucleobase is a cytosine. In some embodiments, the second nucleobase is a deaminated cytosine, or a uracil. In some embodiments, the third nucleobase is a guanine. In some embodiments, the fourth nucleobase is an adenine. In some embodiments, the first nucleobase is a cytosine, the second nucleobase is a deaminated cytosine, or a uracil, the third nucleobase is a guanine, and the fourth nucleobase is an adenine. In some embodiments, the method results in less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the method further comprises replacing the second nucleobase with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited base pair (e.g., C:G→T:A). In some embodiments, the fifth nucleobase is a thymine. In some embodiments, at least 5% of the intended basepairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended basepairs are edited.

0530In some embodiments, the ratio of intended products to unintended products in the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments, the ratio of intended point mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand (nicked strand) is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the base editor comprises a Cas9 domain. In some embodiments, the first base is cytosine, and the second base is not a G, C, A, or T. In some embodiments, the second base is uracil. In some embodiments, the first base is cytosine. In some embodiments, the second base is not a G, C, A, or T. In some embodiments, the second base is uracil. In some embodiments, the base editor inhibits base escision repair of the edited strand. In some embodiments, the base editor protects or binds the non-edited strand. In some embodiments, the base editor comprises UGI activity. In some embodiments, the base editor comprises nickase activity. In some embodiments, the intended edited basepair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited basepair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair is within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the method is performed using any of the base editors provided herein. In some embodiments, a target window is a deamination window.

0531In some embodiments, the disclosure provides methods for editing a nucleotide. In some embodiments, the disclosure provides a method for editing a nucleobase pair of a double-stranded DNA sequence. In some embodiments, the method comprises a) contacting a target region of the double-stranded DNA sequence with a complex comprising a base editor and a guide nucleic acid (e.g., gRNA), where the target region comprises a target nucleobase pair, b) inducing strand separation of said target region, c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase, d) cutting no more than one strand of said target region, wherein a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase, and the second nucleobase is replaced with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited basepair, wherein the efficiency of generating the intended edited basepair is at least 5%. It should be appreciated that in some embodiments, step b is omitted. In some embodiments, at least 5% of the intended basepairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended basepairs are edited. In some embodiments, the method causes less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the ratio of intended product to unintended products at the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments, the ratio of intended point mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the first base is cytosine. In some embodiments, the second nucleobase is not G, C, A, or T. In some embodiments, the second base is uracil. In some embodiments, the base editor inhibits base escision repair of the edited strand. In some embodiments, the base editor protects or binds the non-edited strand. In some embodiments, the nucleobase editor comprises UGI activity. In some embodiments, the nucleobase edit comprises nickase activity. In some embodiments, the intended edited basepair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited basepair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair occurs within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the nucleobase editor is any one of the base editors provided herein.

Pharmaceutical Compositions

0532In some embodiments, any of the fusion proteins, gRNAs, and/or complexes described herein are provided as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any of the fusion proteins provided herein. In some embodiments, the pharmaceutical composition comprises any of the complexes provided herein. In some embodiments, the pharmaceutical composition comprises a ribonucleoprotein complex comprising an RNA-guided nuclease (e.g., Cas9) that forms a complex with a gRNA and a cationic lipid. In some embodiments pharmaceutical composition comprises a gRNA, a nucleic acid programmable DNA binding protein, a cationic lipid, and a pharmaceutically acceptable excipient. Pharmaceutical compositions may optionally comprise one or more additional therapeutically active substances.

0533In some embodiments, compositions provided herein are administered to a subject, for example, to a human subject, in order to effect a targeted genomic modification within the subject. In some embodiments, cells are obtained from the subject and contacted with a any of the pharmaceutical compositions provided herein. In some embodiments, cells removed from a subject and contacted ex vivo with a pharmaceutical composition are re-introduced into the subject, optionally after the desired genomic modification has been effected or detected in the cells. Methods of delivering pharmaceutical compositions comprising nucleases are known, and are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated by reference herein in their entireties. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals or organisms of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, domesticated animals, pets, and commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.

0534Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.

0535Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. <i>Remington's The Science and Practice of Pharmacy, </i>21^stEdition, A. R. Gennaro (Lippincott, Williams &amp; Wilkins, Baltimore, Md., 2006; incorporated in its entirety herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. See also PCT application PCT/US2010/055131 (Publication number WO2011053982 A8, filed Nov. 2, 2010), incorporated in its entirety herein by reference, for additional suitable methods, reagents, excipients and solvents for producing pharmaceutical compositions comprising a nuclease. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

0536In some embodiments, compositions in accordance with the present invention may be used for treatment of any of a variety of diseases, disorders, and/or conditions, including but not limited to one or more of the following: autoimmune disorders (e.g. diabetes, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis); inflammatory disorders (e.g. arthritis, pelvic inflammatory disease); infectious diseases (e.g. viral infections (e.g., HIV, HCV, RSV), bacterial infections, fungal infections, sepsis); neurological disorders (e.g. Alzheimer's disease, Huntington's disease; autism; Duchenne muscular dystrophy); cardiovascular disorders (e.g. atherosclerosis, hypercholesterolemia, thrombosis, clotting disorders, angiogenic disorders such as macular degeneration); proliferative disorders (e.g. cancer, benign neoplasms); respiratory disorders (e.g. chronic obstructive pulmonary disease); digestive disorders (e.g. inflammatory bowel disease, ulcers); musculoskeletal disorders (e.g. fibromyalgia, arthritis); endocrine, metabolic, and nutritional disorders (e.g. diabetes, osteoporosis); urological disorders (e.g. renal disease); psychological disorders (e.g. depression, schizophrenia); skin disorders (e.g. wounds, eczema); blood and lymphatic disorders (e.g. anemia, hemophilia); etc.

0000Kits, Vectors, Cells

0537Some aspects of this disclosure provide kits comprising a nucleic acid construct, comprising (a) a nucleotide sequence encoding a Cas9 protein or a Cas9 fusion protein as provided herein; and (b) a heterologous promoter that drives expression of the sequence of (a). In some embodiments, the kit further comprises an expression construct encoding a guide RNA backbone, wherein the construct comprises a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to a target sequence into the guide RNA backbone.

0538Some aspects of this disclosure provide polynucleotides encoding a Cas9 protein of a fusion protein as provided herein. Some aspects of this disclosure provide vectors comprising such polynucleotides. In some embodiments, the vector comprises a heterologous promoter driving expression of polynucleotide.

0539Some aspects of this disclosure provide cells comprising a Cas9 protein, a fusion protein, a nucleic acid molecule encoding the fusion protein, a complex comprise the Cas9 protein and the gRNA, and/or a vector as provided herein.

0540The description of exemplary embodiments of the reporter systems above is provided for illustration purposes only and not meant to be limiting. Additional reporter systems, e.g., variations of the exemplary systems described in detail above, are also embraced by this disclosure.

EXAMPLES

Example 1: Cas9 Deaminase Fusion Proteins

0541A number of Cas9:Deaminase fusion proteins were generated and deaminase activity of the generated fusions was characterized. The following deaminases were tested:

0000Human AID (hAID):

0542<tables id="TABLE-US-00098" num="00098"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 49)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLR</entry></row><row><entry></entry></row><row><entry>NKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG</entry></row><row><entry></entry></row><row><entry>NPYLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNT</entry></row><row><entry></entry></row><row><entry>FVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGLLD</entry></row></tbody></tgroup></table></tables><br /> Human AID-DC (hAID-DC, Truncated Version of hAID with 7-Fold Increased Activity):

0543<tables id="TABLE-US-00099" num="00099"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 50)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLR</entry></row><row><entry></entry></row><row><entry>NKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG</entry></row><row><entry></entry></row><row><entry>NPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNT</entry></row><row><entry></entry></row><row><entry>FVENHERTFKAWEGLHENSVRLSRQLRRILL</entry></row></tbody></tgroup></table></tables><br /> Rat APOBEC1 (rAPOBEC1):

0544<tables id="TABLE-US-00100" num="00100"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 76)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLK</entry></row></tbody></tgroup></table></tables><br /> Human APOBEC1 (hAPOBEC1)

0545<tables id="TABLE-US-00101" num="00101"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 74)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKI</entry></row><row><entry></entry></row><row><entry>WRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAI</entry></row><row><entry></entry></row><row><entry>REFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYY</entry></row><row><entry></entry></row><row><entry>HCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQ</entry></row><row><entry></entry></row><row><entry>NHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR</entry></row></tbody></tgroup></table></tables><br /><i>Petromyzon marinus </i>(Lamprey) CDA1 (pmCDA1):

0546<tables id="TABLE-US-00102" num="00102"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 81)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFW</entry></row><row><entry></entry></row><row><entry>GYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADC</entry></row><row><entry></entry></row><row><entry>AEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNV</entry></row><row><entry></entry></row><row><entry>MVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKIL</entry></row><row><entry></entry></row><row><entry>HTTKSPAV</entry></row></tbody></tgroup></table></tables><br /> Human APOBEC3G (hAPOBEC3G):

0547<tables id="TABLE-US-00103" num="00103"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 60)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLA</entry></row><row><entry></entry></row><row><entry>EDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQH</entry></row><row><entry></entry></row><row><entry>CWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNE</entry></row><row><entry></entry></row><row><entry>PWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAE</entry></row><row><entry></entry></row><row><entry>LCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCI</entry></row><row><entry></entry></row><row><entry>FTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQ</entry></row><row><entry></entry></row><row><entry>PWDGLDEHSQDLSGRLRAILQNQEN</entry></row></tbody></tgroup></table></tables>

0548Deaminase Activity on ssDNA. A USER (Uracil-Specific Excision Reagent) Enzyme-based assay for deamination was employed to test the activity of various deaminases on single-stranded DNA (ssDNA) substrates. USER Enzyme was obtained from New England Biolabs. An ssDNA substrate was provided with a target cytosine residue at different positions. Deamination of the ssDNA cytosine target residue results in conversion of the target cytosine to a uracil. The USER Enzyme excises the uracil base and cleaves the ssDNA backbone at that position, cutting the ssDNA substrate into two shorter fragments of DNA. In some assays, the ssDNA substrate is labeled on one end with a dye, e.g., with a 5′ Cy3 label (the * in the scheme below). Upon deamination, excision, and cleavage of the strand, the substrate can be subjected to electrophoresis, and the substrate and any fragment released from it can be visualized by detecting the label. Where Cy5 is images, only the fragment with the label will be visible via imaging.

0549In one USER Enzyme assay, ssDNA substrates were used that matched the target sequences of the various deaminases tested. Expression cassettes encoding the deaminases tested were inserted into a CMV backbone plasmid that has been used previously in the lab (Addgene plasmid 52970). The deaminase proteins were expressed using a TNT Quick Coupled Transcription/Translation System (Promega) according to the manufacturers recommendations. After 90 min of incubation, 5 mL of lysate was incubated with 5′ Cy3-labeled ssDNA substrate and 1 unit of USER Enzyme (NEB) for 3 hours. The DNA was resolved on a 10% TBE PAGE gel and the DNA was imaged using Cy-dye imaging. A schematic representation of the USER Enzyme assay is shown in <figref idref="DRAWINGS">FIG. 41</figref>.

0550<figref idref="DRAWINGS">FIG. 1</figref> shows the deaminase activity of the tested deaminases on ssDNA substrates, such as Doench 1, Doench 2, G7′ and VEGF Target 2. The rAPOBEC1 enzyme exhibited a substantial amount of deamination on the single-stranded DNA substrate with a canonical NGG PAM, but not with a negative control non-canonical NNN PAM. Cas9 fusion proteins with APOBEC family deaminases were generated. The following fusion architectures were constructed and tested on ssDNA:

0551<tables id="TABLE-US-00104" num="00104"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="280pt" align="left" /><colspec colname="2" colwidth="161pt" align="left" /><tbody valign="top"><row><entry>rAPOBEC1-GGS-dCas9 primary sequence</entry><entry /></row><row><entry>(SEQ ID NO: 149)</entry><entry /></row><row><entry><u style="single">MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTN</u></entry><entry /></row><row><entry></entry></row><row><entry><u style="single">KHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARL</u></entry></row><row><entry></entry></row><row><entry><u style="single">YHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVR</u></entry></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00008" num="00008"><img file="US11268082B2_D0008.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00009" num="00009"><img file="US11268082B2_D0009.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00010" num="00010"><img file="US11268082B2_D0010.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00011" num="00011"><img file="US11268082B2_D0011.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00012" num="00012"><img file="US11268082B2_D0012.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00013" num="00013"><img file="US11268082B2_D0013.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00014" num="00014"><img file="US11268082B2_D0014.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00015" num="00015"><img file="US11268082B2_D0015.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00016" num="00016"><img file="US11268082B2_D0016.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00017" num="00017"><img file="US11268082B2_D0017.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00018" num="00018"><img file="US11268082B2_D0018.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00019" num="00019"><img file="US11268082B2_D0019.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00020" num="00020"><img file="US11268082B2_D0020.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00021" num="00021"><img file="US11268082B2_D0021.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00022" num="00022"><img file="US11268082B2_D0022.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00023" num="00023"><img file="US11268082B2_D0023.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00024" num="00024"><img file="US11268082B2_D0024.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00025" num="00025"><img file="US11268082B2_D0025.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00026" num="00026"><img file="US11268082B2_D0026.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00027" num="00027"><img file="US11268082B2_D0027.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00028" num="00028"><img file="US11268082B2_D0028.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00029" num="00029"><img file="US11268082B2_D0029.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00030" num="00030"><img file="US11268082B2_D0030.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00031" num="00031"><img file="US11268082B2_D0031.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00032" num="00032"><img file="US11268082B2_D0032.tif" /></chemistry></entry></row><row><entry>rAPOBEC1-(GGS)₃-dCas9 primary sequence</entry></row><row><entry>(SEQ ID NO: 150)</entry><entry /></row><row><entry><u style="single">MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTN</u></entry><entry /></row><row><entry></entry></row><row><entry><u style="single">KHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARL</u></entry></row><row><entry></entry></row><row><entry><u style="single">YHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVR</u></entry></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00033" num="00033"><img file="US11268082B2_D0033.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00034" num="00034"><img file="US11268082B2_D0034.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00035" num="00035"><img file="US11268082B2_D0035.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00036" num="00036"><img file="US11268082B2_D0036.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00037" num="00037"><img file="US11268082B2_D0037.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00038" num="00038"><img file="US11268082B2_D0038.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00039" num="00039"><img file="US11268082B2_D0039.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00040" num="00040"><img file="US11268082B2_D0040.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00041" num="00041"><img file="US11268082B2_D0041.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00042" num="00042"><img file="US11268082B2_D0042.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00043" num="00043"><img file="US11268082B2_D0043.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00044" num="00044"><img file="US11268082B2_D0044.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00045" num="00045"><img file="US11268082B2_D0045.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00046" num="00046"><img file="US11268082B2_D0046.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00047" num="00047"><img file="US11268082B2_D0047.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00048" num="00048"><img file="US11268082B2_D0048.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00049" num="00049"><img file="US11268082B2_D0049.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00050" num="00050"><img file="US11268082B2_D0050.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00051" num="00051"><img file="US11268082B2_D0051.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00052" num="00052"><img file="US11268082B2_D0052.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00053" num="00053"><img file="US11268082B2_D0053.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00054" num="00054"><img file="US11268082B2_D0054.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00055" num="00055"><img file="US11268082B2_D0055.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00056" num="00056"><img file="US11268082B2_D0056.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00057" num="00057"><img file="US11268082B2_D0057.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00058" num="00058"><img file="US11268082B2_D0058.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 151)</entry><entry /></row><row><entry><chemistry id="CHEM-US-00059" num="00059"><img file="US11268082B2_D0059.tif" /></chemistry></entry><entry /></row><row><entry><chemistry id="CHEM-US-00060" num="00060"><img file="US11268082B2_D0060.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00061" num="00061"><img file="US11268082B2_D0061.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00062" num="00062"><img file="US11268082B2_D0062.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00063" num="00063"><img file="US11268082B2_D0063.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00064" num="00064"><img file="US11268082B2_D0064.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00065" num="00065"><img file="US11268082B2_D0065.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00066" num="00066"><img file="US11268082B2_D0066.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00067" num="00067"><img file="US11268082B2_D0067.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00068" num="00068"><img file="US11268082B2_D0068.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00069" num="00069"><img file="US11268082B2_D0069.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00070" num="00070"><img file="US11268082B2_D0070.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00071" num="00071"><img file="US11268082B2_D0071.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00072" num="00072"><img file="US11268082B2_D0072.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00073" num="00073"><img file="US11268082B2_D0073.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00074" num="00074"><img file="US11268082B2_D0074.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00075" num="00075"><img file="US11268082B2_D0075.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00076" num="00076"><img file="US11268082B2_D0076.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00077" num="00077"><img file="US11268082B2_D0077.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00078" num="00078"><img file="US11268082B2_D0078.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00079" num="00079"><img file="US11268082B2_D0079.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00080" num="00080"><img file="US11268082B2_D0080.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00081" num="00081"><img file="US11268082B2_D0081.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00082" num="00082"><img file="US11268082B2_D0082.tif" /></chemistry></entry></row><row><entry><u style="single">FDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSIT</u></entry></row><row><entry></entry></row><row><entry><u style="single">WFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMT</u></entry></row><row><entry></entry></row><row><entry><u style="single">EQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQL</u></entry></row><row><entry></entry></row><row><entry><u style="single">TFFTIALQSCHYQRLPPHILWATGLK</u></entry></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00083" num="00083"><img file="US11268082B2_D0083.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 152)</entry><entry /></row><row><entry><chemistry id="CHEM-US-00084" num="00084"><img file="US11268082B2_D0084.tif" /></chemistry></entry><entry /></row><row><entry><chemistry id="CHEM-US-00085" num="00085"><img file="US11268082B2_D0085.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00086" num="00086"><img file="US11268082B2_D0086.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00087" num="00087"><img file="US11268082B2_D0087.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00088" num="00088"><img file="US11268082B2_D0088.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00089" num="00089"><img file="US11268082B2_D0089.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00090" num="00090"><img file="US11268082B2_D0090.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00091" num="00091"><img file="US11268082B2_D0091.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00092" num="00092"><img file="US11268082B2_D0092.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00093" num="00093"><img file="US11268082B2_D0093.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00094" num="00094"><img file="US11268082B2_D0094.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00095" num="00095"><img file="US11268082B2_D0095.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00096" num="00096"><img file="US11268082B2_D0096.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00097" num="00097"><img file="US11268082B2_D0097.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00098" num="00098"><img file="US11268082B2_D0098.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00099" num="00099"><img file="US11268082B2_D0099.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00100" num="00100"><img file="US11268082B2_D0100.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00101" num="00101"><img file="US11268082B2_D0101.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00102" num="00102"><img file="US11268082B2_D0102.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00103" num="00103"><img file="US11268082B2_D0103.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00104" num="00104"><img file="US11268082B2_D0104.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00105" num="00105"><img file="US11268082B2_D0105.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00106" num="00106"><img file="US11268082B2_D0106.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00107" num="00107"><img file="US11268082B2_D0107.tif" /></chemistry></entry></row><row><entry><u style="single">HEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCP</u></entry></row><row><entry></entry></row><row><entry><u style="single">NTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSG</u></entry></row><row><entry></entry></row><row><entry><u style="single">VTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNIL</u></entry></row><row><entry></entry></row><row><entry><u style="single">RRKQPQLTFFTIALQSCHYQRLPPHILWATGLK</u></entry></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00108" num="00108"><img file="US11268082B2_D0108.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 153)</entry><entry /></row><row><entry><u style="single">MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTN</u></entry><entry /></row><row><entry></entry></row><row><entry><u style="single">KHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARL</u></entry></row><row><entry></entry></row><row><entry><u style="single">YHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVR</u></entry></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00109" num="00109"><img file="US11268082B2_D0109.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00110" num="00110"><img file="US11268082B2_D0110.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00111" num="00111"><img file="US11268082B2_D0111.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00112" num="00112"><img file="US11268082B2_D0112.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00113" num="00113"><img file="US11268082B2_D0113.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00114" num="00114"><img file="US11268082B2_D0114.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00115" num="00115"><img file="US11268082B2_D0115.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00116" num="00116"><img file="US11268082B2_D0116.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00117" num="00117"><img file="US11268082B2_D0117.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00118" num="00118"><img file="US11268082B2_D0118.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00119" num="00119"><img file="US11268082B2_D0119.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00120" num="00120"><img file="US11268082B2_D0120.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00121" num="00121"><img file="US11268082B2_D0121.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00122" num="00122"><img file="US11268082B2_D0122.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00123" num="00123"><img file="US11268082B2_D0123.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00124" num="00124"><img file="US11268082B2_D0124.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00125" num="00125"><img file="US11268082B2_D0125.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00126" num="00126"><img file="US11268082B2_D0126.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00127" num="00127"><img file="US11268082B2_D0127.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00128" num="00128"><img file="US11268082B2_D0128.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00129" num="00129"><img file="US11268082B2_D0129.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00130" num="00130"><img file="US11268082B2_D0130.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00131" num="00131"><img file="US11268082B2_D0131.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00132" num="00132"><img file="US11268082B2_D0132.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00133" num="00133"><img file="US11268082B2_D0133.tif" /></chemistry></entry></row></tbody></tgroup></table></tables>

0552<figref idref="DRAWINGS">FIG. 2</figref> shows that the N-terminal deaminase fusions showed significant activity on the single stranded DNA substrates. For this reason, only the N-terminal architecture was chosen for further experiments.

0553<figref idref="DRAWINGS">FIG. 3</figref> illustrates double stranded DNA substrate binding by deaminase-dCas9:sgRNA complexes. A number of double stranded deaminase substrate sequences were generated. The sequences are provided below. The structures according to <figref idref="DRAWINGS">FIG. 3</figref> are identified in these sequences (36 bp: underlined, sgRNA target sequence: bold; PAM: boxed; 21 bp: italicized). All substrates were labeled with a 5′-Cy3 label:

0554<tables id="TABLE-US-00105" num="00105"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="259pt" align="right" /><colspec colname="2" colwidth="182pt" align="left" /><tbody valign="top"><row><entry>(SEQ ID NO: 85)</entry><entry /></row><row><entry>2: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGTC</u><b>CCGCGGATTTATTTATTTA</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00134" num="00134"><img file="US11268082B2_D0134.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 86)</entry><entry /></row><row><entry>3: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCT</u><b>TCCGCGGATTTATTTATTT</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00135" num="00135"><img file="US11268082B2_D0135.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 87)</entry><entry /></row><row><entry>4: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC</u><b>TTCCGCGGATTTATTTATT</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00136" num="00136"><img file="US11268082B2_D0136.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 88)</entry><entry /></row><row><entry>5: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC</u><b>ATTCCGCGGATTTATTTAT</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00137" num="00137"><img file="US11268082B2_D0137.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 89)</entry><entry /></row><row><entry>6: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC</u><b>TATTCCGCGGATTTATTTA</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00138" num="00138"><img file="US11268082B2_D0138.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 90)</entry><entry /></row><row><entry>7: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC</u><b>TTATTCCGCGGATTTATTT</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00139" num="00139"><img file="US11268082B2_D0139.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 91)</entry><entry /></row><row><entry>8: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC</u><b>ATTATTCCGCGGATTTATT</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00140" num="00140"><img file="US11268082B2_D0140.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 92)</entry><entry /></row><row><entry>9: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC</u><b>TATTATTCCGCGGATTTAT</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00141" num="00141"><img file="US11268082B2_D0141.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 93)</entry><entry /></row><row><entry>10: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC</u><b>ATTATATTCCGCGGATTT</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00142" num="00142"><img file="US11268082B2_D0142.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 94)</entry><entry /></row><row><entry>11: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC</u><b>TATTATATTCCGCGGATT</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00143" num="00143"><img file="US11268082B2_D0143.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 95)</entry><entry /></row><row><entry>12: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC</u><b>TTATTATATTCCGCGGAT</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00144" num="00144"><img file="US11268082B2_D0144.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 96)</entry><entry /></row><row><entry>13: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC</u><b>ATTATTATATTCCGCGGA</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00145" num="00145"><img file="US11268082B2_D0145.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 97)</entry><entry /></row><row><entry>14: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC</u><b>TATTATTATATTCCGCGG</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00146" num="00146"><img file="US11268082B2_D0146.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 98)</entry><entry /></row><row><entry>15: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC</u><b>ATTATTATTATTACCGCG</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00147" num="00147"><img file="US11268082B2_D0147.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 99)</entry><entry /></row><row><entry>18: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC</u><b>ATTATTATTATTATTACCG</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00148" num="00148"><img file="US11268082B2_D0148.tif" /></chemistry></entry></row><row><entry>“-“: </entry></row><row><entry>(SEQ ID NO: 100)</entry><entry /></row><row><entry><chemistry id="CHEM-US-00149" num="00149"><img file="US11268082B2_D0149.tif" /></chemistry></entry><entry /></row><row><entry><chemistry id="CHEM-US-00150" num="00150"><img file="US11268082B2_D0150.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 101)</entry><entry /></row><row><entry>8U: <u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTGTAG</u><b>ATTATTATCUGCGGATTTAT</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00151" num="00151"><img file="US11268082B2_D0151.tif" /></chemistry></entry></row><row><entry>*In all substrates except for “8U”, the top strand in FIG. 3</entry></row><row><entry>is the complement of the sequence specified here. In the case</entry></row><row><entry>of “8U”, there is a “G” opposite the U.</entry></row></tbody></tgroup></table></tables>

0555<figref idref="DRAWINGS">FIG. 4</figref> shows the results of a double stranded DNA Deamination Assay. The fusions were expressed and purified with an N-terminal His6 tag via both Ni-NTA and sepharose chromatography. In order to assess deamination on dsDNA substrates, the various dsDNA substrates shown on the previous slide were incubated at a 1:8 dsDNA:fusion protein ratio and incubated at 37° C. for 2 hours. Once the dCas9 portion of the fusion binds to the DNA it blocks access of the USER enzyme to the DNA. Therefore, the fusion proteins were denatured following the incubation and the dsDNA was purified on a spin column, followed by incubation for 45 min with the USER Enzyme and resolution of the resulting DNA substrate and substrate fragments on a 10% TBE-urea gel.

0556<figref idref="DRAWINGS">FIG. 5</figref> demonstrates that Cas9 fusions can target positions 3-11 of double-stranded DNA target sequences (numbered according to the schematic in <figref idref="DRAWINGS">FIG. 3</figref>). Upper Gel: 1 μM rAPOBEC1-GGS-dCas9, 125 nM dsDNA, 1 eq sgRNA. Mid Gel: 1 μM rAPOBEC1-(GGS)₃-dCas9, 125 nM dsDNA, 1 eq sgRNA. Lower Gel: 1.85 μM rAPOBEC1-XTEN-dCas9, 125 nM dsDNA, 1 eq sgRNA. Based on the data from these gels, positions 3-11 (according to the numbering in <figref idref="DRAWINGS">FIG. 3</figref>) are sufficiently exposed to the activity of the deaminase to be targeted by the fusion proteins tested. Access of the deaminase to other positions is most likely blocked by the dCas9 protein.

0557The data further indicates that a linker of only 3 amino acids (GGS) is not optimal for allowing the deaminase to access the single stranded portion of the DNA. The 9 amino acid linker [(GGS)₃] (SEQ ID NO: 610) and the more structured 16 amino acid linker (XTEN) allow for more efficient deamination.

0558<figref idref="DRAWINGS">FIG. 6</figref> demonstrates that the correct guide RNA, e.g., the correct sgRNA, is required for deaminase activity. The gel shows that fusing the deaminase to dCas9, the deaminase enzyme becomes sequence specific (e.g., using the fusion with an eGFP sgRNA results in no deamination), and also confers the capacity to the deaminase to deaminate dsDNA. The native substrate of the deaminase enzyme is ssDNA, and no deamination occurred when no sgRNA was added. This is consistent with reported knowledge that APOBEC deaminase by itself does not deaminate dsDNA. The data indicates that Cas9 opens the double-stranded DNA helix within a short window, exposing single-stranded DNA that is then accessible to the APOBEC deaminase for cytidine deamination. The sgRNA sequences used are provided below. sequences (36 bp: underlined, sgRNA target sequence: bold; PAM: boxed; 21 bp: italicized) DNA sequence 8:

0559<tables id="TABLE-US-00106" num="00106"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 102)</entry></row><row><entry><u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC</u><b>ATTATTCCGCGGAT</b></entry></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00152" num="00152"><img file="US11268082B2_D0152.tif" /></chemistry></entry></row></tbody></tgroup></table></tables><br /> Correct sgRNA Sequence (Partial 3′ Sequence):

0560<tables id="TABLE-US-00107" num="00107"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 103)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>5′-<b>AUUAUUCCGCGGAUUUAUUU</b>GUUUUAGAGCUAG . . . -3′</entry></row></tbody></tgroup></table></tables><br /> eGFP sgRNA Sequence (Partial 3′-Sequence:

0561<tables id="TABLE-US-00108" num="00108"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 104)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>5′-<b>CGUAGGCCAGGGUGGUCACG</b>GUUUUAGAGCUAG . . . -3′</entry></row></tbody></tgroup></table></tables>

Example 2: Deamination of DNA Target Sequence

0562Exemplary deamination targets. The dCas9:deaminase fusion proteins described herein can be delivered to a cell in vitro or ex vivo or to a subject in vivo and can be used to effect C to T or G to A transitions when the target nucleotide is in positions 3-11 with respect to a PAM. Exemplary deamination targets include, without limitation, the following: CCR5 truncations: any of the codons encoding Q93, Q102, Q186, R225, W86, or Q261 of CCR5 can be deaminated to generate a STOP codon, which results in a nonfunctional truncation of CCR5 with applications in HIV treatment. APOE4 mutations: mutant codons encoding C11R and C57R mutant APOE4 proteins can be deaminated to revert to the wild-type amino acid with applications in Alzheimer's treatment. eGFP truncations: any of the codons encoding Q158, Q184, Q185 can be deaminated to generate a STOP codon, or the codon encoding M1 can be deaminated to encode I, all of which result in loss of eGFP fluorescence, with applications in reporter systems. eGFP restoration: a mutant codon encoding T65A or Y66C mutant GFP, which does not exhibit substantial fluorescence, can be deaminated to restore the wild-type amino acid and confer fluorescence. PIK3CA mutation: a mutant codon encoding K111E mutant PIK3CA can be deaminated to restore the wild-type amino acid residue with applications in cancer. CTNNB1 mutation: a mutant codon encoding T41A mutant CTNNB1 can be deaminated to restore the wild-type amino acid residue with applications in cancer. HRAS mutation: a mutant codon encoding Q61R mutant HRAS can be deaminated to restore the wild-type amino acid residue with applications in cancer. P53 mutations: any of the mutant codons encoding Y163C, Y236C, or N239D mutant p53 can be deaminated to encode the wild type amino acid sequence with applications in cancer. The feasibility of deaminating these target sequences in double-stranded DNA is demonstrated in <figref idref="DRAWINGS">FIGS. 7 and 8</figref>. <figref idref="DRAWINGS">FIG. 7</figref> illustrates the mechanism of target DNA binding of in vivo target sequences by deaminase-dCas9:sgRNA complexes.

0563<figref idref="DRAWINGS">FIG. 8</figref> shows successful deamination of exemplary disease-associated target sequences. Upper Gel: CCR5 Q93: coding strand target in pos. 10 (potential off-targets at positions 2, 5, 6, 8, 9); CCR5 Q102: coding strand target in pos. 9 (potential off-targets at positions 1, 12, 14); CCR5 Q186: coding strand target in pos. 9 (potential off-targets at positions 1, 5, 15); CCR5 R225: coding strand target in pos. 6 (no potential off-targets); eGFP Q158: coding strand target in pos. 5 (potential off-targets at positions 1, 13, 16); eGFP Q184/185: coding strand target in pos. 4 and 7 (potential off-targets at positions 3, 12, 14, 15, 16, 17, 18); eGFP M1: template strand target in pos. 12 (potential off-targets at positions 2, 3, 7, 9, 11) (targets positions 7 and 9 to small degree); eGFP T65A: template strand target in pos. 7 (potential off-targets at positions 1, 8, 17); PIK3CA K111E: template strand target in pos. 2 (potential off-targets at positions 5, 8, 10, 16, 17); PIK3CA K111E: template strand target in pos. 13 (potential off-targets at positions 11, 16, 19) X. Lower Gel: CCR5 W86: template strand target in pos. 2 and 3 (potential off-targets at positions 1, 13) X; APOE4 C11R: coding strand target in pos. 11 (potential off-targets at positions 7, 13, 16, 17); APOE4 C57R: coding strand target in pos. 5) (potential off-targets at positions 7, 8, 12); eGFP Y66C: template strand target in pos. 11 (potential off-targets at positions 1, 4, 6, 8, 9, 16); eGFP Y66C: template strand target in pos. 3 (potential off-targets at positions 1, 8, 17); CCR5 Q261: coding strand target in pos. 10 (potential off-targets at positions 3, 5, 6, 9, 18); CTNNB1 T41A: template strand target in pos. 7 (potential off-targets at positions 1, 13, 15, 16) X; HRAS Q61R: template strand target in pos. 6 (potential off-targets at positions 1, 2, 4, 5, 9, 10, 13); p53 Y163C: template strand target in pos. 6 (potential off-targets at positions 2, 13, 14); p53 Y236C: template strand target in pos. 8 (potential off-targets at positions 2, 4); p53 N239D: template strand target in pos. 4 (potential off-targets at positions 6, 8). Exemplary DNA sequences of disease targets are provided below (PAMs (5′-NGG-3′) and target positions are boxed):

0564<tables id="TABLE-US-00109" num="00109"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="266pt" align="left" /><colspec colname="2" colwidth="175pt" align="left" /><tbody valign="top"><row><entry>CCR5 Q93: 5′-Cy3-</entry><entry /></row><row><entry>(SEQ ID NO: 105)</entry><entry /></row><row><entry><u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTAACTAT</u><b>GCTGCCGCC</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00153" num="00153"><img file="US11268082B2_D0153.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 106)</entry><entry /></row><row><entry>CCR5 Q102: 5′-Cy3-</entry><entry /></row><row><entry><u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTAAAATA</u><b>CAATGTGT</b></entry></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00154" num="00154"><img file="US11268082B2_D0154.tif" /></chemistry></entry></row><row><entry>CCR5 Q186: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 107)</entry><entry /></row><row><entry><u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATTTTC</u><b>CATACAGT</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00155" num="00155"><img file="US11268082B2_D0155.tif" /></chemistry></entry></row><row><entry>CCR5 R225: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 108)</entry><entry /></row><row><entry><chemistry id="CHEM-US-00156" num="00156"><img file="US11268082B2_D0156.tif" /></chemistry></entry><entry /></row><row><entry><chemistry id="CHEM-US-00157" num="00157"><img file="US11268082B2_D0157.tif" /></chemistry></entry></row><row><entry>CCR5 W86: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 109)</entry><entry /></row><row><entry><chemistry id="CHEM-US-00158" num="00158"><img file="US11268082B2_D0158.tif" /></chemistry></entry><entry /></row><row><entry><chemistry id="CHEM-US-00159" num="00159"><img file="US11268082B2_D0159.tif" /></chemistry></entry></row><row><entry>CCR5 Q261: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 110)</entry><entry /></row><row><entry><u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATCCTG</u><b>AACACCTT</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00160" num="00160"><img file="US11268082B2_D0160.tif" /></chemistry></entry></row><row><entry>APOE4 C11R: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 111)</entry><entry /></row><row><entry><u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTAGACAT</u><b>GGAGGAC</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00161" num="00161"><img file="US11268082B2_D0161.tif" /></chemistry></entry></row><row><entry>APOE4 C57R: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 112)</entry><entry /></row><row><entry><chemistry id="CHEM-US-00162" num="00162"><img file="US11268082B2_D0162.tif" /></chemistry></entry><entry /></row><row><entry><chemistry id="CHEM-US-00163" num="00163"><img file="US11268082B2_D0163.tif" /></chemistry></entry></row><row><entry>eGFP Q158: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 113)</entry><entry /></row><row><entry><chemistry id="CHEM-US-00164" num="00164"><img file="US11268082B2_D0164.tif" /></chemistry></entry><entry /></row><row><entry><chemistry id="CHEM-US-00165" num="00165"><img file="US11268082B2_D0165.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 114)</entry><entry /></row><row><entry><chemistry id="CHEM-US-00166" num="00166"><img file="US11268082B2_D0166.tif" /></chemistry></entry><entry /></row><row><entry><chemistry id="CHEM-US-00167" num="00167"><img file="US11268082B2_D0167.tif" /></chemistry></entry></row><row><entry>eGFP M1: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 115)</entry><entry /></row><row><entry><u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTACCTCG</u><b>CCCTTGCTCA</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00168" num="00168"><img file="US11268082B2_D0168.tif" /></chemistry></entry></row><row><entry>eGFP T65A: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 116)</entry><entry /></row><row><entry><chemistry id="CHEM-US-00169" num="00169"><img file="US11268082B2_D0169.tif" /></chemistry></entry><entry /></row><row><entry><chemistry id="CHEM-US-00170" num="00170"><img file="US11268082B2_D0170.tif" /></chemistry></entry></row><row><entry>eGFP Y66C: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 117)</entry><entry /></row><row><entry><u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTAAAGCA</u><b>CTGCACTC</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00171" num="00171"><img file="US11268082B2_D0171.tif" /></chemistry></entry></row><row><entry>eGFP Y66C: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 118)</entry><entry /></row><row><entry><chemistry id="CHEM-US-00172" num="00172"><img file="US11268082B2_D0172.tif" /></chemistry></entry><entry /></row><row><entry><chemistry id="CHEM-US-00173" num="00173"><img file="US11268082B2_D0173.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 119)</entry><entry /></row><row><entry><chemistry id="CHEM-US-00174" num="00174"><img file="US11268082B2_D0174.tif" /></chemistry></entry><entry /></row><row><entry><chemistry id="CHEM-US-00175" num="00175"><img file="US11268082B2_D0175.tif" /></chemistry></entry></row><row><entry>(SEQ ID NO: 120)</entry><entry /></row><row><entry>PIK3CA K111E: 5′-Cy3-<u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTATTCTC</u><b>GATTG</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00176" num="00176"><img file="US11268082B2_D0176.tif" /></chemistry></entry></row><row><entry>CTNNB1 T41A: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 121)</entry><entry /></row><row><entry><u style="single">GTAGGTAGTTAGGATGAATGGAAGGTTGGTAAGGAG</u><b>CTGTGG</b></entry><entry /></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00177" num="00177"><img file="US11268082B2_D0177.tif" /></chemistry></entry></row><row><entry>HRAS Q61R: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 122)</entry><entry /></row><row><entry><chemistry id="CHEM-US-00178" num="00178"><img file="US11268082B2_D0178.tif" /></chemistry></entry><entry /></row><row><entry><chemistry id="CHEM-US-00179" num="00179"><img file="US11268082B2_D0179.tif" /></chemistry></entry></row><row><entry>p53 Y163C: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 123)</entry><entry /></row><row><entry><chemistry id="CHEM-US-00180" num="00180"><img file="US11268082B2_D0180.tif" /></chemistry></entry><entry /></row><row><entry><chemistry id="CHEM-US-00181" num="00181"><img file="US11268082B2_D0181.tif" /></chemistry></entry></row><row><entry>p53 Y236C: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 124)</entry><entry /></row><row><entry><chemistry id="CHEM-US-00182" num="00182"><img file="US11268082B2_D0182.tif" /></chemistry></entry><entry /></row><row><entry><chemistry id="CHEM-US-00183" num="00183"><img file="US11268082B2_D0183.tif" /></chemistry></entry></row><row><entry>p53 N239D: 5′-Cy3-</entry></row><row><entry>(SEQ ID NO: 125)</entry><entry /></row><row><entry><chemistry id="CHEM-US-00184" num="00184"><img file="US11268082B2_D0184.tif" /></chemistry></entry><entry /></row><row><entry><chemistry id="CHEM-US-00185" num="00185"><img file="US11268082B2_D0185.tif" /></chemistry></entry></row></tbody></tgroup></table></tables>

Example 3: Uracil Glycosylase Inhibitor Fusion Improves Deamination Efficiency

0565Direct programmable nucleobase editing efficiencies in mammalian cells by dCas9:deaminase fusion proteins can be improved significantly by fusing a uracil glycosylase inhibitor (UGI) to the dCas9:deaminase fusion protein.

0566<figref idref="DRAWINGS">FIG. 9</figref> shows in vitro C→T editing efficiencies in human HEK293 cells using rAPOBEC1-XTEN-dCas9:

0567<tables id="TABLE-US-00110" num="00110"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="259pt" align="left" /><colspec colname="2" colwidth="182pt" align="left" /><tbody valign="top"><row><entry><chemistry id="CHEM-US-00186" num="00186"><img file="US11268082B2_D0186.tif" /></chemistry></entry><entry /></row><row><entry>(SEQ ID NO: 126)</entry><entry /></row><row><entry><u style="single">MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTN</u></entry><entry /></row><row><entry></entry></row><row><entry><u style="single">KHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLY</u></entry></row><row><entry></entry></row><row><entry><u style="single">HHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRL</u></entry></row><row><entry></entry></row><row><entry><u style="single">YVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK</u><b>SGSETPGTS</b></entry></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00187" num="00187"><img file="US11268082B2_D0187.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00188" num="00188"><img file="US11268082B2_D0188.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00189" num="00189"><img file="US11268082B2_D0189.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00190" num="00190"><img file="US11268082B2_D0190.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00191" num="00191"><img file="US11268082B2_D0191.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00192" num="00192"><img file="US11268082B2_D0192.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00193" num="00193"><img file="US11268082B2_D0193.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00194" num="00194"><img file="US11268082B2_D0194.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00195" num="00195"><img file="US11268082B2_D0195.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00196" num="00196"><img file="US11268082B2_D0196.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00197" num="00197"><img file="US11268082B2_D0197.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00198" num="00198"><img file="US11268082B2_D0198.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00199" num="00199"><img file="US11268082B2_D0199.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00200" num="00200"><img file="US11268082B2_D0200.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00201" num="00201"><img file="US11268082B2_D0201.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00202" num="00202"><img file="US11268082B2_D0202.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00203" num="00203"><img file="US11268082B2_D0203.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00204" num="00204"><img file="US11268082B2_D0204.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00205" num="00205"><img file="US11268082B2_D0205.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00206" num="00206"><img file="US11268082B2_D0206.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00207" num="00207"><img file="US11268082B2_D0207.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00208" num="00208"><img file="US11268082B2_D0208.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00209" num="00209"><img file="US11268082B2_D0209.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00210" num="00210"><img file="US11268082B2_D0210.tif" /></chemistry></entry></row></tbody></tgroup></table></tables><br /> Protospacer Sequences were as Follows:

0568<tables id="TABLE-US-00111" num="00111"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 127)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="77pt" align="left" /><colspec colname="2" colwidth="140pt" align="left" /><tbody valign="top"><row><entry>EMX1:</entry><entry><chemistry id="CHEM-US-00211" num="00211"><img file="US11268082B2_D0211.tif" /></chemistry></entry></row><row><entry /><entry>(SEQ ID NO: 128)</entry></row><row><entry>FANCF:</entry><entry><chemistry id="CHEM-US-00212" num="00212"><img file="US11268082B2_D0212.tif" /></chemistry></entry></row><row><entry /><entry>(SEQ ID NO: 129)</entry></row><row><entry>HEK293 site 2:</entry><entry><chemistry id="CHEM-US-00213" num="00213"><img file="US11268082B2_D0213.tif" /></chemistry></entry></row><row><entry /><entry>(SEQ ID NO: 130)</entry></row><row><entry>HEK293 site 3:</entry><entry><chemistry id="CHEM-US-00214" num="00214"><img file="US11268082B2_D0214.tif" /></chemistry></entry></row><row><entry /><entry>(SEQ ID NO: 735)</entry></row><row><entry>HEK293 site 4:</entry><entry><chemistry id="CHEM-US-00215" num="00215"><img file="US11268082B2_D0215.tif" /></chemistry></entry></row><row><entry /><entry>(SEQ ID NO: 132)</entry></row><row><entry>RNF2:</entry><entry><chemistry id="CHEM-US-00216" num="00216"><img file="US11268082B2_D0216.tif" /></chemistry></entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>*PAMs are boxed, C residues within target window</entry></row><row><entry>(positions 3-11) are numbered and bolded.</entry></row></tbody></tgroup></table></tables>

0569<figref idref="DRAWINGS">FIG. 10</figref> demonstrates that C→T editing efficiencies on the same protospacer sequences in HEK293T cells are greatly enhanced when a UGI domain is fused to the rAPOBEC1:dCas9 fusion protein.

0570<tables id="TABLE-US-00112" num="00112"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="259pt" align="left" /><colspec colname="2" colwidth="182pt" align="left" /><tbody valign="top"><row><entry><chemistry id="CHEM-US-00217" num="00217"><img file="US11268082B2_D0217.tif" /></chemistry></entry><entry /></row><row><entry>(SEQ ID NO: 133)</entry><entry /></row><row><entry><u style="single">MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTN</u></entry><entry /></row><row><entry></entry></row><row><entry><u style="single">KHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLY</u></entry></row><row><entry></entry></row><row><entry><u style="single">HHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRL</u></entry></row><row><entry></entry></row><row><entry><u style="single">YVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK</u><b>SGSETPGTS</b></entry></row><row><entry></entry></row><row><entry><chemistry id="CHEM-US-00218" num="00218"><img file="US11268082B2_D0218.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00219" num="00219"><img file="US11268082B2_D0219.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00220" num="00220"><img file="US11268082B2_D0220.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00221" num="00221"><img file="US11268082B2_D0221.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00222" num="00222"><img file="US11268082B2_D0222.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00223" num="00223"><img file="US11268082B2_D0223.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00224" num="00224"><img file="US11268082B2_D0224.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00225" num="00225"><img file="US11268082B2_D0225.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00226" num="00226"><img file="US11268082B2_D0226.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00227" num="00227"><img file="US11268082B2_D0227.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00228" num="00228"><img file="US11268082B2_D0228.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00229" num="00229"><img file="US11268082B2_D0229.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00230" num="00230"><img file="US11268082B2_D0230.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00231" num="00231"><img file="US11268082B2_D0231.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00232" num="00232"><img file="US11268082B2_D0232.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00233" num="00233"><img file="US11268082B2_D0233.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00234" num="00234"><img file="US11268082B2_D0234.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00235" num="00235"><img file="US11268082B2_D0235.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00236" num="00236"><img file="US11268082B2_D0236.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00237" num="00237"><img file="US11268082B2_D0237.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00238" num="00238"><img file="US11268082B2_D0238.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00239" num="00239"><img file="US11268082B2_D0239.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00240" num="00240"><img file="US11268082B2_D0240.tif" /></chemistry></entry></row><row><entry><chemistry id="CHEM-US-00241" num="00241"><img file="US11268082B2_D0241.tif" /></chemistry></entry></row><row><entry><u style="single"><b>LMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVICIDSNGE</b></u></entry></row><row><entry></entry></row><row><entry><u style="single"><b>NKIKML</b></u>SGGS<i>PKKKRKV</i></entry></row></tbody></tgroup></table></tables>

0571The percentages in <figref idref="DRAWINGS">FIGS. 9 and 10</figref> are shown from sequencing both strands of the target sequence. Because only one of the strands is a substrate for deamination, the maximum possible deamination value in this assay is 50%. Accordingly, the deamination efficiency is double the percentages shown in the tables. E.g., a value of 50% relates to deamination of 100% of double-stranded target sequences.

0572When a uracil glycosylase inhibitor (UGI) was fused to the dCas9:deaminase fusion protein (e.g., rAPOBEC1-XTEN-dCas9-[UGI]-NLS), a significant increase in editing efficiency in cells was observed. This result indicates that in mammalian cells, the DNA repair machinery that cuts out the uracil base in a U:G base pair is a rate-limiting process in DNA editing. Tethering UGI to the dVas9:deaminase fusion proteins greatly increases editing yields.

0573Without UGI, typical editing efficiencies in human cells were in the ˜2-14% yield range (<figref idref="DRAWINGS">FIG. 9</figref> and <figref idref="DRAWINGS">FIG. 10</figref>, “XTEN” entries). With UGI (<figref idref="DRAWINGS">FIG. 10</figref>, “UGI” entries) the editing was observed in the ˜6-40% range. Using a UGI fusion is thus more efficient than the current alternative method of correcting point mutations via HDR, which also creates an excess of indels in addition to correcting the point mutation. No indels resulting from treatment with the cas9:deaminase:UGI fusions were observed.

Example 4: Direct, Programmable Conversion of a Target Nucleotide in Genomic DNA without Double-Stranded DNA Cleavage

0574Current genome-editing technologies introduce double-stranded DNA breaks at a target locus of interest as the first step to gene correction.^39,40Although most genetic diseases arise from mutation of a single nucleobase to a different nucleobase, current approaches to revert such changes are very inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus as a consequence of the cellular response to double-stranded DNA breaks.^39,40Reported herein is the development of nucleobase editing, a new strategy for genome editing that enables the direct conversion of one target nucleobase into another in a programmable manner, without requiring double-stranded DNA backbone cleavage. Fusions of CRISPR/Cas9 were engineered and the cytidine deaminase enzyme APOBEC1 that retain the ability to be programmed with a guide RNA, do not induce double-stranded DNA breaks, and mediate the direct conversion of cytidine to uracil, thereby effecting a C→T (or G→A) substitution following DNA replication, DNA repair, or transcription if the template strand is targeted. The resulting “nucleobase editors” convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease in vitro. In four transformed human and murine cell lines, second- and third-generation nucleobase editors that fuse uracil glycosylase inhibitor (UGI), and that use a Cas9 nickase targeting the non-edited strand, respectively, can overcome the cellular DNA repair response to nucleobase editing, resulting in permanent correction of up to 37% or (˜15-75%) of total cellular DNA in human cells with minimal (typically ≤1%) indel formation. In contrast, canonical Cas9-mediated HDR on the same targets yielded an average of 0.7% correction with 4% indel formation. Nucleobase editors were used to revert two oncogenic p53 mutations into wild-type alleles in human breast cancer and lymphoma cells, and to convert an Alzheimer's Disease associated Arg codon in ApoE4 into a non-disease-associated Cys codon in mouse astrocytes. Base editing expands the scope and efficiency of genome editing of point mutations.

0575The clustered regularly interspaced short palindromic repeat (CRISPR) system is a prokaryotic adaptive immune system that has been adapted to mediate genome engineering in a variety of organisms and cell lines.⁴¹CRISPR/Cas9 protein-RNA complexes localize to a target DNA sequence through base pairing with a guide RNA, and natively create a DNA double-stranded break (DSB) at the locus specified by the guide RNA. In response to DSBs, endogenous DNA repair processes mostly result in random insertions or deletions (indels) at the site of DNA cleavage through non-homologous end joining (NHEJ). In the presence of a homologous DNA template, the DNA surrounding the cleavage site can be replaced through homology-directed repair (HDR). When simple disruption of a disease-associated gene is sufficient (for example, to treat some gain-of-function diseases), targeted DNA cleavage followed by indel formation can be effective. For most known genetic diseases, however, correction of a point mutation in the target locus, rather than stochastic disruption of the gene, is needed to address or study the underlying cause of the disease.⁶⁸

0576Motivated by this need, researchers have invested intense effort to increase the efficiency of HDR and suppress NHEJ. For example, a small-molecule inhibitor of ligase IV, an essential enzyme in the NHEJ pathway, has been shown to increase HDR efficiency.^42,43However, this strategy is challenging in post-mitotic cells, which typically down-regulate HDR, and its therapeutic relevance is limited by the potential risks of inhibiting ligase IV in non-target cells. Enhanced HDR efficiency can also be achieved by the timed delivery of Cas9-guide RNA complexes into chemically synchronized cells, as HDR efficiency is highly cell-cycle dependent.⁴⁴Such an approach, however, is limited to research applications in cell culture since synchronizing cells is highly disruptive. Despite these developments, current strategies to replace point mutations using HDR in most contexts are very inefficient (typically ˜0.1 to 5%),^{42,43,45,46,75}especially in unmodified, non-dividing cells. In addition, HDR competes with NHEJ during the resolution of double-stranded breaks, and indels are generally more abundant outcomes than gene replacement. These observations highlight the need to develop alternative approaches to install specific modifications in genomic DNA that do not rely on creating double-stranded DNA breaks. A small-molecule inhibitor of ligase IV, an essential enzyme in the NHEJ pathway, has been shown to increase HDR efficiency.^42,43However, this strategy is challenging in post-mitotic cells, which typically down-regulate HDR, and its therapeutic relevance is limited by the potential risks of inhibiting ligase IV in non-target cells. Enhanced HDR efficiency can also be achieved by the timed delivery of Cas9-guide RNA complexes into chemically synchronized cells, as HDR efficiency is highly cell-cycle dependent.⁴⁴Such an approach, however, is limited to research applications in cell culture since synchronizing cells is highly disruptive. In some cases, it is possible to design HDR templates such that the product of successful HDR contains mutations in the PAM sequence and therefore is no longer a substrate for subsequent Cas9 modification, increasing the overall yield of HDR products,⁷⁵although such an approach imposes constraints on the product sequences. Recently, this strategy has been coupled to the use of ssDNA donors that are complementary to the non-target strand and high-efficiency ribonucleoprotein (RNP) delivery to substantially increase the efficiency of HDR, but even in these cases the ratio of HDR to NHEJ outcomes is relatively low (&lt;2).⁸³

0577It was envisioned that direct catalysis of the conversion of one nucleobase to another at a programmable target locus without requiring DNA backbone cleavage could increase the efficiency of gene correction relative to HDR without introducing undesired random indels at the locus of interest. Catalytically dead Cas9 (dCas9), which contains Asp10Ala and His840Ala mutations that inactivate its nuclease activity, retains its ability to bind DNA in a guide RNA-programmed manner but does not cleave the DNA backbone.^16,47In principle, conjugation of dCas9 with an enzymatic or chemical catalyst that mediates the direct conversion of one nucleobase to another could enable RNA-programmed nucleobase editing. The deamination of cytosine (C) is catalyzed by cytidine deaminases²⁹and results in uracil (U), which has the base pairing properties of thymine (T). dCas9 was fused to cytidine deaminase enzymes in order to test their ability to convert C to U at a guide RNA-specified DNA locus. Most known cytidine deaminases operate on RNA, and the few examples that are known to accept DNA require single-stranded DNA.⁴⁸Recent studies on the dCas9-target DNA complex reveal that at least nine nucleotides of the displaced DNA strand are unpaired upon formation of the Cas9:guide RNA:DNA “R-loop” complex.¹²Indeed, in the structure of the Cas9 R-loop complex the first 11 nucleotides of the protospacer on the displaced DNA strand are disordered, suggesting that their movement is not highly restricted.⁷⁶It has also been speculated that Cas9 nickase-induced mutations at cytosines in the non-template strand might arise from their accessibility by cellular cytidine deaminase enzymes.⁷⁷Recent studies on the dCas9-target DNA complex have revealed that at least 26 bases on the non-template strand are unpaired when Cas9 binds to its target DNA sequence.⁴⁹It was reasoned that a subset of this stretch of single-stranded DNA in the R-loop might serve as a substrate for a dCas9-tethered cytidine deaminase to effect direct, programmable conversion of C to U in DNA (<figref idref="DRAWINGS">FIG. 11A</figref>).

0578Four different cytidine deaminase enzymes (hAID, hAPOBEC3G, rAPOBEC1, and pmCDA1) were expressed in a mammalian cell lysate-derived in vitro transcription-translation system and evaluated for ssDNA deamination. Of the four enzymes, rAPOBEC1 showed the highest deaminase activity under the tested conditions and was chosen for dCas9 fusion experiments (<figref idref="DRAWINGS">FIG. 36A</figref>). Although appending rAPOBEC1 to the C-terminus of dCas9 abolishes deaminase activity, fusion to the N-terminus of dCas9 preserves deaminase activity on ssDNA at a level comparable to that of the unfused enzyme. Four rAPOBEC1-dCas9 fusions were expressed and purified with linkers of different length and composition (<figref idref="DRAWINGS">FIG. 36B</figref>), and evaluated each fusion for single guide RNA (sgRNA)-programmed dsDNA deamination in vitro (<figref idref="DRAWINGS">FIGS. 11A to 11C</figref> and <figref idref="DRAWINGS">FIGS. 15A to 15D</figref>).

0579Efficient, sequence-specific, sgRNA-dependent C to U conversion was observed in vitro (<figref idref="DRAWINGS">FIGS. 11A to 11C</figref>). Conversion efficiency was greatest using rAPOBEC1-dCas9 linkers over nine amino acids in length. The number of positions susceptible to deamination (the deamination “activity window”) increases with linker length was extended from three to 21 amino acids (<figref idref="DRAWINGS">FIGS. 36C to 36F</figref><b>15</b>A to <b>15</b>D). The 16-residue XTEN linker⁵⁰was found to offer a promising balance between these two characteristics, with an efficient deamination window of approximately five nucleotides, from positions 4 to 8 within the protospacer, counting the end distal to the protospacer-adjacent motif (PAM) as position 1. The rAPOBEC1-XTEN-dCas9 protein served as the first-generation nucleobase editor (NBE1).

0580Elected were seven mutations relevant to human disease that in theory could be corrected by C to T nucleobase editing, synthesized double-stranded DNA 80-mers of the corresponding sequences, and assessed the ability of NBE1 to correct these mutations in vitro (<figref idref="DRAWINGS">FIGS. 16A to 16B</figref>). NBE1 yielded products consistent with efficient editing of the target C, or of at least one C within the activity window when multiple Cs were present, in six of these seven targets in vitro, with an average apparent editing efficiency of 44% (<figref idref="DRAWINGS">FIGS. 16A to 16B</figref>). In the three cases in which multiple Cs were present within the deamination window, evidence of deamination of some or all of these cytosines was observed. In only one of the seven cases tested were substantial yields of edited product observed (<figref idref="DRAWINGS">FIGS. 16A to 16B</figref>). Although the preferred sequence context for APOBEC1 substrates is reported to be CC or TC,⁵¹it was anticipated that the increased effective molarity of the deaminase and its single-stranded DNA substrate mediated by dCas9 binding to the target locus may relax this restriction. To illuminate the sequence context generality of NBE1, its ability to edit a 60-mer double-stranded DNA oligonucleotide containing a single fixed C at position 7 within the protospacer was assayed, as well as all 36 singly mutated variants in which protospacer bases 1-6 and 8-13 were individually varied to each of the other three bases. Each of these 37 sequences were treated with 1.9 μM NBE1, 1.9 μM of the corresponding sgRNA, and 125 nM DNA for 2 h, similar to standard conditions for in vitro Cas9 assays⁵². High-throughput DNA sequencing (HTS) revealed 50 to 80% C to U conversion of targeted strands (25 to 40% of total sequence reads arising from both DNA strands, one of which is not a substrate for NBE1) (<figref idref="DRAWINGS">FIG. 12A</figref>). The nucleotides surrounding the target C had little effect on editing efficiency was independent of sequence context unless the base immediately 5′ of the target C is a G, in which case editing efficiency was substantially lower (<figref idref="DRAWINGS">FIGS. 12A to 12B</figref>). NBE1 activity in vitro was assessed on all four NC motifs at positions 1 through 8 within the protospacer (<figref idref="DRAWINGS">FIGS. 12A to 12B</figref>). In general, NBE1 activity on substrates was observed to follow the order TC≥CC≥AC>GC, with maximum editing efficiency achieved when the target C is at or near position 7. In addition, it was observed that the nucleobase editor is highly processive, and will efficiently convert most of all Cs to Us on the same DNA strand within the 5-base activity window (<figref idref="DRAWINGS">FIG. 17</figref>).

0581While BE1 efficiently processes substrates in a test tube, in cells a tree of possible DNA repair outcomes determines the fate of the initial U:G product of base editing (<figref idref="DRAWINGS">FIG. 29A</figref>). To test the effectiveness of nucleobase editing in human cells, NBE1 codon usage was optimized for mammalian expression, appended a C-terminal nuclear localization sequence (NLS),⁵³and assayed its ability to convert C to T in human cells on 14Cs in six well-studied target sites throughout the human genome (<figref idref="DRAWINGS">FIG. 37A</figref>).⁵⁴The editable Cs were confirmed within each protospacer in vitro by incubating NBE1 with synthetic 80-mers that correspond to the six different genomic sites, followed by HTS (<figref idref="DRAWINGS">FIGS. 13A to 13C</figref>, <figref idref="DRAWINGS">FIG. 29B</figref> and <figref idref="DRAWINGS">FIG. 25</figref>). Next, HEK293T cells were transfected with plasmids encoding NBE1 and one of the six target sgRNAs, allowed three days for nucleobase editing to occur, extracted genomic DNA from the cells, and analyzed the loci by HTS. Although C to T editing in cells at the target locus was observed for all six cases, the efficiency of nucleobase editing was 1.1% to 6.3% or 0.8%-7.7% of total DNA sequences (corresponding to 2.2% to 12.6% of targeted strands), a 6.3-fold to 37-fold or 5-fold to 36-fold decrease in efficiency compared to that of in vitro nucleobase editing (<figref idref="DRAWINGS">FIGS. 13A to 13C</figref>, <figref idref="DRAWINGS">FIG. 29B</figref> and <figref idref="DRAWINGS">FIG. 25</figref>). It was observed that some base editing outside of the typical window of positions 4 to 8 when the substrate C is preceded by a T, which we attribute to the unusually high activity of APOBEC1 for TC substrates.⁴⁸

0582It was asked whether the cellular DNA repair response to the presence of U:G heteroduplex DNA was responsible for the large decrease in nucleobase editing efficiency in cells (<figref idref="DRAWINGS">FIG. 29A</figref>). Uracil DNA glycosylase (UDG) catalyzes removal of U from DNA in cells and initiates base excision repair (BER), with reversion of the U:G pair to a C:G pair as the most common outcome (<figref idref="DRAWINGS">FIG. 29A</figref>).⁵⁵Uracil DNA glycosylase inhibitor (UGI), an 83-residue protein from <i>B. subtilis </i>bacteriophage PBS1, potently blocks human UDG activity (IC₅₀=12 pM).⁵⁶UGI was fused to the C-terminus of NBE1 to create the second-generation nucleobase editor NBE2 and repeated editing assays on all six genomic loci. Editing efficiencies in human cells were on average 3-fold higher with NBE2 than with NBE1, resulting in gene conversion efficiencies of up to 22.8% of total DNA sequenced (up to 45.6% of targeted strands) (<figref idref="DRAWINGS">FIGS. 13A to 13C</figref> and <figref idref="DRAWINGS">FIG. 29B</figref>). To test base editing in human cells, BE1 codon usage was optimized for mammalian expression and appended a C-terminal nuclear localization sequence (NLS).⁵³

0583Similar editing efficiencies were observed when a separate plasmid overexpressing UGI was co-transfected with NBE1 (<figref idref="DRAWINGS">FIGS. 18A to 18H</figref>). However, while the direct fusion of UGI to NBE1 resulted in no significant increase in C to T mutations at monitored non-targeted genomic locations, overexpression of unfused UGI detectably increased the frequency of C to T mutations elsewhere in the genome (<figref idref="DRAWINGS">FIGS. 18A to 18H</figref>). The generality of NBE2-mediated nucleobase editing was confirmed by assessing editing efficiencies on the same six genomic targets in U2OS cells, and observed similar results with those in HEK293T cells (<figref idref="DRAWINGS">FIG. 19</figref>). Importantly, NBE2 typically did not result in any detectable indels (<figref idref="DRAWINGS">FIG. 13C</figref> and <figref idref="DRAWINGS">FIG. 29C</figref>), consistent with the known mechanistic dependence of NHEJ on double-stranded DNA breaks.^{57, 78}Together, these results indicate that conjugating UGI to NBE1 can greatly increase the efficiency of nucleobase editing in human cells.

0584The permanence of nucleobase editing in human cells was confirmed by monitoring editing efficiencies over multiple cell divisions in HEK293T cells at two of the tested genomic loci. Genomic DNA was harvested at two time points: three days after transfection with plasmids expressing NBE2 and appropriate sgRNAs, and after passaging the cells and growing them for four additional days (approximately five subsequent cell divisions). No significant change in editing efficiency was observed between the non-passaged cells (editing observed in 4.6% to 6.6% of targeted strands for three different target Cs) and passaged cells (editing observed in 4.6% to 6.4% of targeted strands for the same three target Cs), confirming that the nucleobase edits became permanent following cell division (<figref idref="DRAWINGS">FIG. 20</figref>). Indels will on rare occasion arise from the processing of U:G lesions by cellular repair processes, which involve single-strand break intermediates that are known to lead to indels.⁸⁴Given that several hundred endogenous U:G lesions are generated every day per human cell from spontaneous cytidine deaminase,⁸⁵it was anticipate that the total indel frequency from U:G lesion repair is unlikely to increase from BE1 or BE2 activity at a single target locus.

0585To further increase the efficiency of nucleobase editing in cells, it was anticipated that nicking the non-edited strand may result in a smaller fraction of edited Us being removed by the cell, since eukaryotic mismatch repair machinery uses strand discontinuity to direct DNA repair to any broken strand of a mismatched duplex (<figref idref="DRAWINGS">FIG. 29A</figref>).^{58, 79, 80}The catalytic His residue was restored at position 840 in the Cas9 HNH domain,^47,59resulting in the third-generation nucleobase editor NBE3 that nicks the non-edited strand containing a G opposite the targeted C, but does not cleave the target strand containing the C. Because NBE3 still contains the Asp10Ala mutation in Cas9, it does not induce double-stranded DNA cleavage. This strategy of nicking the non-edited strand augmented nucleobase editing efficiency in human cells by an additional 1.4- to 4.8-fold relative to NBE2, resulting in up to 36.3% of total DNA sequences containing the targeted C to T conversion on the same six human genomic targets in HEK293T cells (<figref idref="DRAWINGS">FIGS. 13A to 13C</figref> and <figref idref="DRAWINGS">FIG. 29B</figref>). Importantly, only a small frequency of indels, averaging 0.8% (ranging from 0.2% to 1.6% for the six different loci), was observed from NBE3 treatment (<figref idref="DRAWINGS">FIG. 13C</figref>, <figref idref="DRAWINGS">FIG. 29C</figref>, and <figref idref="DRAWINGS">FIG. 34</figref>). In contrast, when cells were treated with wild-type Cas9, sgRNA, and a single-stranded DNA donor template to mediate HDR at three of these loci C to T conversion efficiencies averaging only 0.7% were observed, with much higher relative indel formation averaging 3.9% (<figref idref="DRAWINGS">FIGS. 13A to 13C</figref> and <figref idref="DRAWINGS">FIG. 29C</figref>). The ratio of allele conversion to NHEJ outcomes averaged >1,000 for BE2, 23 for BE3, and 0.17 for wild-type Cas9 (<figref idref="DRAWINGS">FIG. 3<i>c</i></figref>). We confirmed the permanence of base editing in human cells by monitoring editing efficiencies over multiple cell divisions in HEK293T cells at the HEK293 site 3 and 4 genomic loci (<figref idref="DRAWINGS">FIG. 38</figref>). These results collectively establish that nucleobase editing can effect much more efficient targeted single-base editing in human cells than Cas9-mediated HDR, and with much less (NBE3) or no (NBE2) indel formation.

0586Next, the off-target activity of NBE1, NBE2, and NBE3 in human cells was evaluated. The off-target activities of Cas9, dCas9, and Cas9 nickase have been extensively studied (<figref idref="DRAWINGS">FIGS. 23 to 24 and 31 to 33</figref>).^54,60-62Because the sequence preference of rAPOBEC1 has been shown to be independent of DNA bases more than one base from the target C,⁶³consistent with the sequence context independence observed in <figref idref="DRAWINGS">FIGS. 12A to 12B</figref>, it was assumed that potential off-target activity of nucleobase editors arises from off-target Cas9 binding. Since only a fraction of Cas9 off-target sites will have a C within the active window for nucleobase editing, off-target nucleobase editing sites should be a subset of the off-target sites of canonical Cas9 variants. For each of the six sites studied, the top ten known Cas9 off-target loci in human cells that were previously determined using the GUIDE-seq method were sequenced (<figref idref="DRAWINGS">FIGS. 23 to 27 and 31 to 33</figref>).^{54, 61}Detectable off-target nucleobase editing at only a subset (16/34, 47% for NBE1 and NBE2, and 17/34, 50% for NBE3) of known dCas9 off-target loci was observed. In all cases, the off-target base-editing substrates contained a C within the five-base target window. In general, off-target C to T conversion paralleled off-target Cas9 nuclease-mediated genome modification frequencies (<figref idref="DRAWINGS">FIGS. 23 to 27</figref>). Also monitored were C to T conversions at 2,500 distinct cytosines surrounding the six on-target and 34 off-target loci tested, representing a total of 14,700,000 sequence reads derived from approximately 1.8×10⁶cells, and observed no detectable increase in C to T conversions at any of these other sites upon NBE1, NBE2, or NBE3 treatment compared to that of untreated cells (<figref idref="DRAWINGS">FIG. 28</figref>). Taken together, these findings suggest that off-target substrates of nucleobase editors include a subset of Cas9 off-target substrates, and that nucleobase editors in human cells do not induce untargeted C to T conversion throughout the genome at levels that can be detected by the methods used here. No substantial change was observed in editing efficiency between non-passaged HEK293T cells (editing observed in 1.8% to 2.6% of sequenced strands for the three target Cs with BE2, and 6.2% to 14.3% with BE3) and cells that had undergone approximately five cell divisions after base editing (editing observed in 1.9% to 2.3% of sequenced strands for the same target Cs with BE2, and 6.4% to 14.5% with BE3), confirming that base edits in these cells are durable (Extended Data <figref idref="DRAWINGS">FIG. 6</figref>).

0587Finally, the potential of nucleobase editing to correct three disease-relevant mutations in mammalian cells was tested. The apolipoprotein E gene variant APOE4 encodes two Arg residues at amino acid positions 112 and 158, and is the largest and most common genetic risk factor for late-onset Alzheimer's disease.⁶⁴ApoE variants with Cys residues in positions 112 or 158, including APOE2 (Cys112/Cys158), APOE3 (Cys112/Arg158), and APOE3′ (Arg112/Cys158) have been shown⁶⁵or are presumed⁸¹to confer substantially lower Alzheimer's disease risk than APOE4. Encouraged by the ability of NBE1 to convert APOE4 to APOE3′ in vitro (<figref idref="DRAWINGS">FIGS. 16A to 16B</figref>), this conversion was attempted in immortalized mouse astrocytes in which the endogenous murine APOE gene has been replaced by human APOE4 (Taconic). DNA encoding NBE3 and an appropriate sgRNA was delivered into these astrocytes by nucleofection (nucleofection efficiency of 25%), extracted genomic DNA from all treated cells two days later, and measured editing efficiency by HTS. Conversion of Arg158 to Cys158 was observed in 58-75% of total DNA sequencing reads (44% of nucleofected astrocytes) (<figref idref="DRAWINGS">FIGS. 14A to 14C</figref> and <figref idref="DRAWINGS">FIG. 30A</figref>). Also observed was 36-50% editing of total DNA at the third position of codon 158 and 38-55% editing of total DNA at the first position of Leu159, as expected since all three of these Cs are within the active nucleobase editing window. However, neither of the other two C→T conversions results in a change in the amino acid sequence of the ApoE3′ protein since both TGC and TGT encode Cys, and both CTG and TTG encode Leu. From >1,500,000 sequencing reads derived from 1×10⁶cells evidence of 1.7% indels at the targeted locus following NBE3 treatment was observed (<figref idref="DRAWINGS">FIG. 35</figref>). In contrast, identical treatment of astrocytes with wt Cas9 and donor ssDNA resulted in 0.1-0.3% APOE4 correction and 26-40% indels at the targeted locus, efficiencies consistent with previous reports of single-base correction using Cas9 and HDR^45,75(<figref idref="DRAWINGS">FIG. 30A</figref> and <figref idref="DRAWINGS">FIG. 40A</figref>). Astrocytes treated identically but with an sgRNA targeting the VEGFA locus displayed no evidence of APOE4 base editing (<figref idref="DRAWINGS">FIG. 34</figref> and <figref idref="DRAWINGS">FIG. 40A</figref>). These results demonstrate how nucleobase editors can effect precise, single-amino acid changes in the coding sequence of a protein as the major product of editing, even when their processivity results in more than one nucleotide change in genomic DNA. The off-target activities of Cas9, dCas9, and Cas9 nickase have been extensively studied.^54,60-62In general, off-target C to T conversions by BE1, BE2, and BE3 paralleled off-target Cas9 nuclease-mediated genome modification frequencies.

0588The dominant-negative p53 mutations Tyr163Cys and Asn239Asp are strongly associated with several types of cancer.^66-67Both of these mutations can be corrected by a C to T conversion on the template strand (<figref idref="DRAWINGS">FIGS. 16A to 16B</figref>). A human breast cancer cell line homozygous for the p53 Tyr163Cys mutation (HCC1954 cells) was nucleofected with DNA encoding NBE3 and an sgRNA programmed to correct Tyr163Cys. Because the nucleofection efficiency of HCC1954 cells was <10%, a plasmid expressing IRFP was co-nucleofected into these cells to enable isolation of nucleofected cells by fluorescence-activated cell sorting two days after treatment. HTS of genomic DNA revealed correction of the Tyr163Cys mutation in 7.6% of nucleofected HCC1954 cells (<figref idref="DRAWINGS">FIG. 30B</figref> and <figref idref="DRAWINGS">FIG. 40A to 40B</figref>). Also nucleofected was a human lymphoma cell line that is heterozygous for p53 Asn239Asp (ST486 cells) with DNA encoding NBE2 and an sgRNA programmed to correct Asn239Asp with 92% nucleofection efficiency). Correction of the Asn239Asp mutation was observed in 11% of treated ST486 cells (12% of nucleofected ST486 cells). Consistent with the findings in HEK cells, no indels were observed from the treatment of ST486 cells with NBE2, and 0.6% indel formation from the treatment of HCC1954 cells with NBE3. No other DNA changes within at least 50 base pairs of both sides of the protospacer were detected at frequencies above that of untreated controls out of >2,000,000 sequencing reads derived from 2×10⁵cells (<figref idref="DRAWINGS">FIGS. 14A to 14C</figref>, <figref idref="DRAWINGS">FIG. 30B</figref>). These results collectively represent the conversion of three disease-associated alleles in genomic DNA into their wild-type forms with an efficiency and lack of other genome modification events that is, to our knowledge, not currently achievable using other methods.

0589To illuminate the potential relevance of nucleobase editors to address human genetic diseases, the NCBI ClinVar database⁶⁸was searched for known genetic diseases that could in principle be corrected by this approach. ClinVar was filtered by first examining only single nucleotide polymorphisms (SNPs), then removing any nonpathogenic variants. Out of the 24,670 pathogenic SNPs, 3,956 are caused by either a T to C, or an A to G, substitution. This list was further filtered to only include variants with a nearby NGG PAM that would position the SNP within the deamination activity window, resulting in 1,089 clinically relevant pathogenic gene variants that could in principle be corrected by the nucleobase editors described here (<figref idref="DRAWINGS">FIG. 21</figref>).

0590In some embodiments, any of the base editors provided herein may be used to treat a disease or disorder. For example, any base editors provided herein may be used to correct one or more mutations associated with any of the diseases or disorders provided herein. Exemplary diseases or disorders that may be treated include, without limitation, 3-Methylglutaconic aciduria type 2, 46,XY gonadal dysgenesis, 4-Alpha-hydroxyphenylpyruvate hydroxylase deficiency, 6-pyruvoyl-tetrahydropterin synthase deficiency, achromatopsia, Acid-labile subunit deficiency, Acrodysostosis, acroerythrokeratoderma, ACTH resistance, ACTH-independent macronodular adrenal hyperplasia, Activated PI3K-delta syndrome, Acute intermittent porphyria, Acute myeloid leukemia, Adams-Oliver syndrome 1/5/6, Adenylosuccinate lyase deficiency, Adrenoleukodystrophy, Adult neuronal ceroid lipofuscinosis, Adult onset ataxia with oculomotor apraxia, Advanced sleep phase syndrome, Age-related macular degeneration, Alagille syndrome, Alexander disease, Allan-Herndon-Dudley syndrome, Alport syndrome, X-linked recessive, Alternating hemiplegia of childhood, Alveolar capillary dysplasia with misalignment of pulmonary veins, Amelogenesis imperfecta, Amyloidogenic transthyretin amyloidosis, Amyotrophic lateral sclerosis, Anemia (nonspherocytic hemolytic, due to G6PD deficiency), Anemia (sideroblastic, pyridoxine-refractory, autosomal recessive), Anonychia, Antithrombin III deficiency, Aortic aneurysm, Aplastic anemia, Apolipoprotein C2 deficiency, Apparent mineralocorticoid excess, Aromatase deficiency, Arrhythmogenic right ventricular cardiomyopathy, Familial hypertrophic cardiomyopathy, Hypertrophic cardiomyopathy, Arthrogryposis multiplex congenital, Aspartylglycosaminuria, Asphyxiating thoracic dystrophy, Ataxia with vitamin E deficiency, Ataxia (spastic), Atrial fibrillation, Atrial septal defect, atypical hemolytic-uremic syndrome, autosomal dominant CD11C+/CD1C+ dendritic cell deficiency, Autosomal dominant progressive external ophthalmoplegia with mitochondrial DNA deletions, Baraitser-Winter syndrome, Bartter syndrome, Basa ganglia calcification, Beckwith-Wiedemann syndrome, Benign familial neonatal seizures, Benign scapuloperoneal muscular dystrophy, Bernard Soulier syndrome, Beta thalassemia intermedia, Beta-D-mannosidosis, Bietti crystalline corneoretinal dystrophy, Bile acid malabsorption, Biotinidase deficiency, Borjeson-Forssman-Lehmann syndrome, Boucher Neuhauser syndrome, Bowen-Conradi syndrome, Brachydactyly, Brown-Vialetto-Van laere syndrome, Brugada syndrome, Cardiac arrhythmia, Cardiofaciocutaneous syndrome, Cardiomyopathy, Carnevale syndrome, Carnitine palmitoyltransferase II deficiency, Carpenter syndrome, Cataract, Catecholaminergic polymorphic ventricular tachycardia, Central core disease, Centromeric instability of chromosomes 1,9 and 16 and immunodeficiency, Cerebral autosomal dominant arteriopathy, Cerebro-oculo-facio-skeletal syndrome, Ceroid lipofuscinosis, Charcot-Marie-Tooth disease, Cholestanol storage disease, Chondrocalcinosis, Chondrodysplasia, Chronic progressive multiple sclerosis, Coenzyme Q10 deficiency, Cohen syndrome, Combined deficiency of factor V and factor VIII, Combined immunodeficiency, Combined oxidative phosphorylation deficiency, Combined partial 17-alpha-hydroxylase/17,20-lyase deficiency, Complement factor d deficiency, Complete combined 17-alpha-hydroxylase/17,20-lyase deficiency, Cone-rod dystrophy, Congenital contractural arachnodactyly, Congenital disorder of glycosylation, Congenital lipomatous overgrowth, Neoplasm of ovary, PIK3CA Related Overgrowth Spectrum, Congenital long QT syndrome, Congenital muscular dystrophy, Congenital muscular hypertrophy-cerebral syndrome, Congenital myasthenic syndrome, Congenital myopathy with fiber type disproportion, Eichsfeld type congenital muscular dystrophy, Congenital stationary night blindness, Corneal dystrophy, Cornelia de Lange syndrome, Craniometaphyseal dysplasia, Crigler Najjar syndrome, Crouzon syndrome, Cutis laxa with osteodystrophy, Cyanosis, Cystic fibrosis, Cystinosis, Cytochrome-c oxidase deficiency, Mitochondrial complex I deficiency, D-2-hydroxyglutaric aciduria, Danon disease, Deafness with labyrinthine aplasia microtia and microdontia (LAMM), Deafness, Deficiency of acetyl-CoA acetyltransferase, Deficiency of ferroxidase, Deficiency of UDPglucose-hexose-1-phosphate uridylyltransferase, Dejerine-Sottas disease, Desbuquois syndrome, DFNA, Diabetes mellitus type 2, Diabetes-deafness syndrome, Diamond-Blackfan anemia, Diastrophic dysplasia, Dihydropteridine reductase deficiency, Dihydropyrimidinase deficiency, Dilated cardiomyopathy, Disseminated atypical mycobacterial infection, Distal arthrogryposis, Distal hereditary motor neuronopathy, Donnai Barrow syndrome, Duchenne muscular dystrophy, Becker muscular dystrophy, Dyschromatosis universalis hereditaria, Dyskeratosis congenital, Dystonia, Early infantile epileptic encephalopathy, Ehlers-Danlos syndrome, Eichsfeld type congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, Enamel-renal syndrome, Epidermolysis bullosa dystrophica inversa, Epidermolysis bullosa herpetiformis, Epilepsy, Episodic ataxia, Erythrokeratodermia variabilis, Erythropoietic protoporphyria, Exercise intolerance, Exudative vitreoretinopathy, Fabry disease, Factor V deficiency, Factor VII deficiency, Factor xiii deficiency, Familial adenomatous polyposis, breast cancer, ovarian cancer, cold urticarial, chronic infantile neurological, cutaneous and articular syndrome, hemiplegic migraine, hypercholesterolemia, hypertrophic cardiomyopathy, hypoalphalipoproteinemia, hypokalemia-hypomagnesemia, juvenile gout, hyperlipoproteinemia, visceral amyloidosis, hypophosphatemic vitamin D refractory rickets, FG syndrome, Fibrosis of extraocular muscles, Finnish congenital nephrotic syndrome, focal epilepsy, Focal segmental glomerulosclerosis, Frontonasal dysplasia, Frontotemporal dementia, Fructose-biphosphatase deficiency, Gamstorp-Wohlfart syndrome, Ganglioside sialidase deficiency, GATA-1-related thrombocytopenia, Gaucher disease, Giant axonal neuropathy, Glanzmann thrombasthenia, Glomerulocystic kidney disease, Glomerulopathy, Glucocorticoid resistance, Glucose-6-phosphate transport defect, Glutaric aciduria, Glycogen storage disease, Gorlin syndrome, Holoprosencephaly, GRACILE syndrome, Haemorrhagic telangiectasia, Hemochromatosis, Hemoglobin H disease, Hemolytic anemia, Hemophagocytic lymphohistiocytosis, Carcinoma of colon, Myhre syndrome, leukoencephalopathy, Hereditary factor IX deficiency disease, Hereditary factor VIII deficiency disease, Hereditary factor XI deficiency disease, Hereditary fructosuria, Hereditary Nonpolyposis Colorectal Neoplasm, Hereditary pancreatitis, Hereditary pyropoikilocytosis, Elliptocytosis, Heterotaxy, Heterotopia, Histiocytic medullary reticulosis, Histiocytosis-lymphadenopathy plus syndrome, HNSHA due to aldolase A deficiency, Holocarboxylase synthetase deficiency, Homocysteinemia, Howel-Evans syndrome, Hydatidiform mole, Hypercalciuric hypercalcemia, Hyperimmunoglobulin D, Mevalonic aciduria, Hyperinsulinemic hypoglycemia, Hyperkalemic Periodic Paralysis, Paramyotonia congenita of von Eulenburg, Hyperlipoproteinemia, Hypermanganesemia, Hypermethioninemia, Hyperphosphatasemia, Hypertension, hypomagnesemia, Hypobetalipoproteinemia, Hypocalcemia, Hypogonadotropic hypogonadism, Hypogonadotropic hypogonadism, Hypohidrotic ectodermal dysplasia, Hyper-IgM immunodeficiency, Hypohidrotic X-linked ectodermal dysplasia, Hypomagnesemia, Hypoparathyroidism, Idiopathic fibrosing alveolitis, Immunodeficiency, Immunoglobulin A deficiency, Infantile hypophosphatasia, Infantile Parkinsonism-dystonia, Insulin-dependent diabetes mellitus, Intermediate maple syrup urine disease, Ischiopatellar dysplasia, Islet cell hyperplasia, Isolated growth hormone deficiency, Isolated lutropin deficiency, Isovaleric acidemia, Joubert syndrome, Juvenile polyposis syndrome, Juvenile retinoschisis, Kallmann syndrome, Kartagener syndrome, Kugelberg-Welander disease, Lattice corneal dystrophy, Leber congenital amaurosis, Leber optic atrophy, Left ventricular noncompaction, Leigh disease, Mitochondrial complex I deficiency, Leprechaunism syndrome, Arthrogryposis, Anterior horn cell disease, Leukocyte adhesion deficiency, Leukodystrophy, Leukoencephalopathy, Ovarioleukodystrophy, L-ferritin deficiency, Li-Fraumeni syndrome, Limb-girdle muscular dystrophy-dystroglycanopathy, Loeys-Dietz syndrome, Long QT syndrome, Macrocephaly/autism syndrome, Macular corneal dystrophy, Macular dystrophy, Malignant hyperthermia susceptibility, Malignant tumor of prostate, Maple syrup urine disease, Marden Walker like syndrome, Marfan syndrome, Marie Unna hereditary hypotrichosis, Mast cell disease, Meconium ileus, Medium-chain acyl-coenzyme A dehydrogenase deficiency, Melnick-Fraser syndrome, Mental retardation, Merosin deficient congenital muscular dystrophy, Mesothelioma, Metachromatic leukodystrophy, Metaphyseal chondrodysplasia, Methemoglobinemia, methylmalonic aciduria, homocystinuria, Microcephaly, chorioretinopathy, lymphedema, Microphthalmia, Mild non-PKU hyperphenylalanemia, Mitchell-Riley syndrome, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency, Mitochondrial complex I deficiency, Mitochondrial complex III deficiency, Mitochondrial myopathy, Mucolipidosis III, Mucopolysaccharidosis, Multiple sulfatase deficiency, Myasthenic syndrome, <i>Mycobacterium tuberculosis</i>, Myeloperoxidase deficiency, Myhre syndrome, Myoclonic epilepsy, Myofibrillar myopathy, Myoglobinuria, Myopathy, Myopia, Myotonia congenital, Navajo neurohepatopathy, Nemaline myopathy, Neoplasm of stomach, Nephrogenic diabetes insipidus, Nephronophthisis, Nephrotic syndrome, Neurofibromatosis, Neutral lipid storage disease, Niemann-Pick disease, Non-ketotic hyperglycinemia, Noonan syndrome, Noonan syndrome-like disorder, Norum disease, Macular degeneration, N-terminal acetyltransferase deficiency, Oculocutaneous albinism, Oculodentodigital dysplasia, Ohdo syndrome, Optic nerve aplasia, Ornithine carbamoyltransferase deficiency, Orofaciodigital syndrome, Osteogenesis imperfecta, Osteopetrosis, Ovarian dysgenesis, Pachyonychia, Palmoplantar keratoderma, nonepidermolytic, Papillon-Lef\xc3\xa8vre syndrome, Haim-Munk syndrome, Periodontitis, Peeling skin syndrome, Pendred syndrome, Peroxisomal fatty acyl-coa reductase 1 disorder, Peroxisome biogenesis disorder, Pfeiffer syndrome, Phenylketonuria, Phenylketonuria, Hyperphenylalaninemia, non-PKU, Pituitary hormone deficiency, <i>Pityriasis rubra </i>pilaris, <i>Polyarteritis nodosa</i>, Polycystic kidney disease, Polycystic lipomembranous osteodysplasia, Polymicrogyria, Pontocerebellar hypoplasia, Porokeratosis, Posterior column ataxia, Primary erythromelalgia, hyperoxaluria, Progressive familial intrahepatic cholestasis, Progressive pseudorheumatoid dysplasia, Propionic acidemia, Pseudohermaphroditism, Pseudohypoaldosteronism, Pseudoxanthoma elasticum-like disorder, Purine-nucleoside phosphorylase deficiency, Pyridoxal 5-phosphate-dependent epilepsy, Renal dysplasia, retinal pigmentary dystrophy, cerebellar ataxia, skeletal dysplasia, Reticular dysgenesis, Retinitis pigmentosa, Usher syndrome, Retinoblastoma, Retinopathy, RRM2B-related mitochondrial disease, Rubinstein-Taybi syndrome, Schnyder crystalline corneal dystrophy, Sebaceous tumor, Severe congenital neutropenia, Severe myoclonic epilepsy in infancy, Severe X-linked myotubular myopathy, onychodysplasia, facial dysmorphism, hypotrichosis, Short-rib thoracic dysplasia, Sialic acid storage disease, Sialidosis, Sideroblastic anemia, Small fiber neuropathy, Smith-Magenis syndrome, Sorsby fundus dystrophy, Spastic ataxia, Spastic paraplegia, Spermatogenic failure, Spherocytosis, Sphingomyelin/cholesterol lipidosis, Spinocerebellar ataxia, Split-hand/foot malformation, Spondyloepimetaphyseal dysplasia, Platyspondylic lethal skeletal dysplasia, Squamous cell carcinoma of the head and neck, Stargardt disease, Sucrase-isomaltase deficiency, Sudden infant death syndrome, Supravalvar aortic stenosis, Surfactant metabolism dysfunction, Tangier disease, Tatton-Brown-rahman syndrome, Thoracic aortic aneurysms and aortic dissections, Thrombophilia, Thyroid hormone resistance, TNF receptor-associated periodic fever syndrome (TRAPS), Tooth agenesis, Torsades de pointes, Transposition of great arteries, Treacher Collins syndrome, Tuberous sclerosis syndrome, Tyrosinase-negative oculocutaneous albinism, Tyrosinase-positive oculocutaneous albinism, Tyrosinemia, UDPglucose-4-epimerase deficiency, Ullrich congenital muscular dystrophy, Bethlem myopathy Usher syndrome, UV-sensitive syndrome, Van der Woude syndrome, popliteal pterygium syndrome, Very long chain acyl-CoA dehydrogenase deficiency, Vesicoureteral reflux, Vitreoretinochoroidopathy, Von Hippel-Lindau syndrome, von Willebrand disease, Waardenburg syndrome, Warsaw breakage syndrome, WFS1-Related Disorders, Wilson disease, Xeroderma pigmentosum, X-linked agammaglobulinemia, X-linked hereditary motor and sensory neuropathy, X-linked severe combined immunodeficiency, and Zellweger syndrome.

0591The development of nucleobase editing advances both the scope and effectiveness of genome editing. The nucleobase editors described here offer researchers a choice of editing with virtually no indel formation (NBE2), or more efficient editing with a low frequency (here, typically &lt;1%) of indel formation (NBE3). That the product of base editing is, by definition, no longer a substrate likely contributes to editing efficiency by preventing subsequent product transformation, which can hamper traditional Cas9 applications. By removing the reliance on double-stranded DNA cleavage and stochastic DNA repair processes that vary greatly by cell state and cell type, nucleobase editing has the potential to expand the type of genome modifications that can be cleanly installed, the efficiency of these modifications, and the type of cells that are amenable to editing. It is likely that recent engineered Cas9 variants^69,70,82or delivery methods⁷¹with improved DNA specificity, as well as Cas9 variants with altered PAM specificities,⁷²can be integrated into this strategy to provide additional nucleobase editors with improved DNA specificity or that can target an even wider range of disease-associated mutations. These findings also suggest that engineering additional fusions of dCas9 with enzymes that catalyze additional nucleobase transformations will increase the fraction of the possible DNA base changes that can be made through nucleobase editing. These results also suggest architectures for the fusion of other DNA-modifying enzymes, including methylases and demathylases, that mau enable additional types of programmable genome and epigenome base editing.

0000Materials and Methods

0592Cloning. DNA sequences of all constructs and primers used in this paper are listed in the Supplementary Sequences. Plasmids containing genes encoding NBE1, NBE2, and NBE3 will be available from Addgene. PCR was performed using VeraSeq ULtra DNA polymerase (Enzymatics), or Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). NBE plasmids were constructed using USER cloning (New England Biolabs). Deaminase genes were synthesized as gBlocks Gene Fragments (Integrated DNA Technologies), and Cas9 genes were obtained from previously reported plasmids.¹⁸Deaminase and fusion genes were cloned into pCMV (mammalian codon-optimized) or pET28b (<i>E. coli </i>codon-optimized) backbones. sgRNA expression plasmids were constructed using site-directed mutagenesis. Briefly, the primers listed in the Supplementary Sequences were 5′ phosphorylated using T4 Polynucleotide Kinase (New England Biolabs) according to the manufacturer's instructions. Next, PCR was performed using Q5 Hot Start High-Fidelity Polymerase (New England Biolabs) with the phosphorylated primers and the plasmid pFYF1320 (EGFP sgRNA expression plasmid) as a template according to the manufacturer's instructions. PCR products were incubated with DpnI (20 U, New England Biolabs) at 37° C. for 1 h, purified on a QIAprep spin column (Qiagen), and ligated using QuickLigase (New England Biolabs) according to the manufacturer's instructions. DNA vector amplification was carried out using Machi competent cells (ThermoFisher Scientific).

0593In Vitro Deaminase Assay on ssDNA.

0594Sequences of all ssDNA substrates are listed in the Supplementary Sequences. All Cy3-labelled substrates were obtained from Integrated DNA Technologies (IDT). Deaminases were expressed in vitro using the TNT T7 Quick Coupled Transcription/Translation Kit (Promega) according to the manufacturer's instructions using 1 μg of plasmid. Following protein expression, 5 μL of lysate was combined with 35 μL of ssDNA (1.8 μM) and USER enzyme (1 unit) in CutSmart buffer (New England Biolabs) (50 mM potassium acetate, 29 mM Trisacetate, 10 mM magnesium acetate, 100 ug/mL BSA, pH 7.9) and incubated at 37° C. for 2 h. Cleaved U-containing substrates were resolved from full-length unmodified substrates on a 10% TBE-urea gel (Bio-Rad).

0595Expression and Purification of His6-rAPOBEC1-Linker-dCas9 Fusions.

0596<i>E. Coli </i>BL21 STAR (DE3)-competent cells (ThermoFisher Scientific) were transformed with plasmids encoding pET28b-His6-rAPOBEC-linker-dCas9 with GGS, (GGS)₃, (SEQ ID NO: 610) XTEN, or (GGS)₇(SEQ ID NO: 610) linkers. The resulting expression strains were grown overnight in Luria-Bertani (LB) broth containing 100 μg/mL of kanamycin at 37° C. The cells were diluted 1:100 into the same growth medium and grown at 37° C. to OD₆₀₀=˜0.6. The culture was cooled to 4° C. over a period of 2 h, and isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added at 0.5 mM to induce protein expression. After ˜16 h, the cells were collected by centrifugation at 4,000 g and resuspended in lysis buffer (50 mM tris(hydroxymethyl)-aminomethane (Tris)-HCl, pH 7.0, 1 M NaCl, 20% glycerol, 10 mM tris(2-carboxyethyl)phosphine (TCEP, Soltec Ventures)). The cells were lysed by sonication (20 s pulse-on. 20 s pulse-off for 8 min total at 6 W output) and the lysate supernatant was isolated following centrifugation at 25,000 g for 15 min. The lysate was incubated with His-Pur nickel-nitriloacetic acid (nickel-NTA) resin (ThermoFisher Scientific) at 4° C. for 1 h to capture the His-tagged fusion protein. The resin was transferred to a column and washed with 40 mL of lysis buffer. The His-tagged fusion protein was eluted in lysis buffer supplemented with 285 mM imidazole, and concentrated by ultrafiltration (Amicon-Millipore, 100-kDa molecular weight cut-off) to 1 mL total volume. The protein was diluted to 20 mL in low-salt purification buffer containing 50 mM tris(hydroxymethyl)-aminomethane (Tris)-HCl, pH 7.0, 0.1 M NaCl, 20% glycerol. 10 mM TCEP and loaded onto SP Sepharose Fast Flow resin (GE Life Sciences). The resin was washed with 40 mL of this low-salt buffer, and the protein eluted with 5 mL, of activity buffer containing 50 mM tris(hydroxymethyl)-aminomethane (Tris)-HCl, pH 7.0. 0.5 M NaCl, 20% glycerol, 10 mM TCEP. The eluted proteins were quantified on a SDSPAGE gel

0597In Vitro Transcription of sgRNAs.

0598Linear DNA fragments containing the T7 promoter followed by the 20-bp sgRNA target sequence were transcribed in vitro using the primers listed in the Supplementary Sequences with the TranscriptAid T7 High Yield Transcription Kit (ThermoFisher Scientific) according to the manufacturer's instructions. sgRNA products were purified using the MEGAclear Kit (ThermoFisher Scientific) according to the manufacturer's instructions and quantified by UV absorbance.

0599Preparation of Cy3-Conjugated dsDNA Substrates.

0600Sequences of 80-nucleotide unlabeled strands are listed in the Supplementary Sequences and were ordered as PAGE-purified oligonucleotides from IDT. The 25-nt Cy3-labeled primer listed in the Supplementary Sequences is complementary to the 3′ end of each 80-nt substrate. This primer was ordered as an HPLC-purified oligonucleotide from IDT. To generate the Cy3-labeled dsDNA substrates, the 80-nt strands (5 μL of a 100 μM solution) were combined with the Cy3-labeled primer (5 L of a 100 μM solution) in NEBuffer 2 (38.25 μL of a 50 mM NaCl, 10 mMTris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.9 solution, New England Biolabs) with dNTPs (0.75 μL of a 100 mM solution) and heated to 95° C. for 5 min, followed by a gradual cooling to 45° C. at a rate of 0.1° C./s. After this annealing period, Klenow exo (5 U, New England Biolabs) was added and the reaction was incubated at 37° C. for 1 h. The solution was diluted with Buffer PB (250 μL, Qiagen) and isopropanol (50 μL) and purified on a QIAprep spin column (Qiagen), eluting with 50 μL of Tris buffer.

0601Deaminase Assay on dsDNA.

0602The purified fusion protein (20 μL of 1.9 μM in activity buffer) was combined with 1 equivalent of appropriate sgRNA and incubated at ambient temperature for 5 min. The Cy3-labeled dsDNA substrate was added to final concentration of 125 nM and the resulting solution was incubated at 37° C. for 2 h. The dsDNA was separated from the fusion by the addition of Buffer PB (100 μL, Qiagen) and isopropanol (25 μL) and purified on a EconoSpin micro spin column (Epoch Life Science), eluting with 20 μL of CutSmart buffer (New England Biolabs). USER enzyme (1 U, New England Biolabs) was added to the purified, edited dsDNA and incubated at 37° C. for 1 h. The Cy3-labeled strand was fully denatured from its complement by combining 5 μL of the reaction solution with 15 μL of a DMSO-based loading buffer (5 mM Tris, 0.5 mM EDTA, 12.5% glycerol, 0.02% bromophenol blue, 0.02% xylene cyan, 80% DMSO). The full-length C-containing substrate was separated from any cleaved, U-containing edited substrates on a 10% TBE-urea gel (Bio-Rad) and imaged on a GE Amersham Typhoon imager.

0603Preparation of In Vitro-Edited dsDNA for High-Throughput Sequencing (HTS).

0604The oligonucleotides listed in the Supplementary Sequences were obtained from IDT. Complementary sequences were combined (5 μL of a 100 μM solution) in Tris buffer and annealed by heating to 95° C. for 5 min, followed by a gradual cooling to 45° C. at a rate of 0.1° C./s to generate 60-bp dsDNA substrates. Purified fusion protein (20 μL of 1.9 μM in activity buffer) was combined with 1 equivalent of appropriate sgRNA and incubated at ambient temperature for 5 min. The 60-mer dsDNA substrate was added to final concentration of 125 nM and the resulting solution was incubated at 37° C. for 2 h. The dsDNA was separated from the fusion by the addition of Buffer PB (100 μL, Qiagen) and isopropanol (25 μL) and purified on a EconoSpin micro spin column (Epoch Life Science), eluting with 20 μL of Tris buffer. The resulting edited DNA (1 μL was used as a template) was amplified by PCR using the HTS primer pairs specified in the Supplementary Sequences and VeraSeq Ultra (Enzymatics) according to the manufacturer's instructions with 13 cycles of amplification. PCR reaction products were purified using RapidTips (Diffinity Genomics), and the purified DNA was amplified by PCR with primers containing sequencing adapters, purified, and sequenced on a MiSeq high-throughput DNA sequencer (Illumina) as previously described.⁷³

0605Cell Culture.

0606HEK293T (ATCC CRL-3216), U2OS (ATCC-HTB-96) and ST486 cells (ATCC) were maintained in Dulbecco's Modified Eagle's Medium plus GlutaMax (ThermoFisher) supplemented with 10% (v/v) fetal bovine serum (FBS) and penicillin/streptomycin (1×, Amresco), at 37° C. with 5% CO₂. HCC1954 cells (ATCC CRL-2338) were maintained in RPMI-1640 medium (ThermoFisher Scientific) supplemented as described above. Immortalized rat astrocytes containing the ApoE4 isoform of the APOE gene (Taconic Biosciences) were cultured in Dulbecco's Modified Eagle's Medium plus GlutaMax (ThermoFisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS) and 200 g/mL Geneticin (ThermoFisher Scientific).

0607Transfections.

0608HEK293T cells were seeded on 48-well collagen-coated BioCoat plates (Corning) and transfected at approximately 85% confluency. Briefly, 750 ng of NBE and 250 ng of sgRNA expression plasmids were transfected using 1.5 μl of LIPOFECTAMINE® 2000 transfection reagent (ThermoFisher Scientific) per well according to the manufacturer's protocol. Astrocytes, U2OS, HCC1954, HEK293T and ST486 cells were transfected using appropriate AMAXA NUCLEOFECTOR™ II programs according to manufacturer's instructions. 40 ng of infrared RFP (Addgene plasmid 45457)⁷⁴was added to the nucleofection solution to assess nucleofection efficiencies in these cell lines. For astrocytes, U2OS, and ST486 cells, nucleofection efficiencies were 25%, 74%, and 92%, respectively. For HCC1954 cells, nucleofection efficiency was &lt;10%. Therefore, following trypsinization, the HCC1954 cells were filtered through a 40 micron strainer (Fisher Scientific), and the nucleofected HCC1954 cells were collected on a Beckman Coulter MoFlo XDP Cell Sorter using the iRFP signal (abs 643 nm, em 670 nm). The other cells were used without enrichment of nucleofected cells.

0609High-Throughput DNA Sequencing of Genomic DNA Samples.

0610Transfected cells were harvested after 3 d and the genomic DNA was isolated using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter) according to the manufacturer's instructions. On-target and off-target genomic regions of interest were amplified by PCR with flanking HTS primer pairs listed in the Supplementary Sequences. PCR amplification was carried out with Phusion high-fidelity DNA polymerase (ThermoFisher) according to the manufacturer's instructions using 5 ng of genomic DNA as a template. Cycle numbers were determined separately for each primer pair as to ensure the reaction was stopped in the linear range of amplification (30, 28, 28, 28, 32, and 32 cycles for EMX1, FANCF, HEK293 site 2, HEK293 site 3, HEK293 site 4, and RNF2 primers, respectively). PCR products were purified using RapidTips (Diffinity Genomics). Purified DNA was amplified by PCR with primers containing sequencing adaptors. The products were gel-purified and quantified using the QUANT-IT™ PicoGreen dsDNA Assay Kit (ThermoFisher) and KAPA Library Quantification Kit-Illumina (KAPA Biosystems). Samples were sequenced on an Illumina MiSeq as previously described.⁷³

0611Data Analysis.

0612Sequencing reads were automatically demultiplexed using MiSeq Reporter (Illumina), and individual FASTQ files were analyzed with a custom Matlab script provided in the Supplementary Notes. Each read was pairwise aligned to the appropriate reference sequence using the Smith-Waterman algorithm. Base calls with a Q-score below 31 were replaced with N's and were thus excluded in calculating nucleotide frequencies. This treatment yields an expected MiSeq base-calling error rate of approximately 1 in 1,000. Aligned sequences in which the read and reference sequence contained no gaps were stored in an alignment table from which base frequencies could be tabulated for each locus.

0613Indel frequencies were quantified with a custom Matlab script shown in the Supplementary Notes using previously described criteria⁷¹. Sequencing reads were scanned for exact matches to two 10-bp sequences that flank both sides of a window in which indels might occur. If no exact matches were located, the read was excluded from analysis. If the length of this indel window exactly matched the reference sequence the read was classified as not containing an indel. If the indel window was two or more bases longer or shorter than the reference sequence, then the sequencing read was classified as an insertion or deletion, respectively.

0614All publications, patents, patent applications, publication, and database entries (e.g., sequence database entries) mentioned herein, e.g., in the Background, Summary, Detailed Description, Examples, and/or References sections, are hereby incorporated by reference in their entirety as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated herein by reference. In case of conflict, the present application, including any definitions herein, will control.

0000Supplementary Sequences

0615Primers Used for Generating sgRNA Transfection Plasmids.

0616rev_sgRNA_plasmid was used in all cases. The pFYF1320 plasmid was used as template as noted in Materials and Methods section. SEQ ID NOs: 187-196 appear from top to bottom below, respectively.

0617<tables id="TABLE-US-00113" num="00113"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="77pt" align="left" /><colspec colname="2" colwidth="238pt" align="left" /><colspec colname="3" colwidth="126pt" align="left" /><tbody valign="top"><row><entry>rev_sgRNA_plasmid</entry><entry>GGTGTTTCGTCCTTTCCACAAG</entry><entry /></row><row><entry></entry></row><row><entry>fxd_p53_Y163C</entry><entry>GCTTGCAGATGGCCATGGCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC</entry></row><row><entry></entry></row><row><entry>fwd_p53_N239D</entry><entry>TGTCACACATGTAGTTGTAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC</entry></row><row><entry></entry></row><row><entry>fwd_APOE4_C158R</entry><entry>GAAGCGCCTGGCAGTGTACCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC</entry></row><row><entry></entry></row><row><entry>fwd_EMX1</entry><entry>GAGTCCGAGCAGAAGAAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC</entry></row><row><entry></entry></row><row><entry>fwd_FANCF</entry><entry>GGAATCCCTTCTGCAGCACCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_2</entry><entry>GAACACAAAGCATAGACTGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_3</entry><entry>GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_4</entry><entry>GGCACTGCGGCTGGAGGTGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC</entry></row><row><entry></entry></row><row><entry>fwd_RNF2</entry><entry>GTCATCTTAGTCATTACCTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC</entry></row></tbody></tgroup></table></tables>

0618Sequences of all ssDNA Substrates Used in In Vitro Deaminase Assays.

0619SEQ ID NOs: 197-199 appear from top to bottom below, respectively.

0620<tables id="TABLE-US-00114" num="00114"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="49pt" align="left" /><colspec colname="2" colwidth="168pt" align="left" /><tbody valign="top"><row><entry>rAPOBEC1</entry><entry>Cy3-ATTATTATTATTCCGCGGATTTATTTATTTATTTA</entry></row><row><entry>substrate</entry><entry>TTTATTT</entry></row><row><entry></entry></row><row><entry>hAID/pmCDA1</entry><entry>Cy3-ATTATTATTATTAGCTATTTATTTATTTATTTATT</entry></row><row><entry>substrate</entry><entry>TATTT</entry></row><row><entry></entry></row><row><entry>hAPOBEC3G</entry><entry>Cy3-ATTATTATTATTCCCGGATTTATTTATTTATTTAT</entry></row><row><entry>substrate</entry><entry>TTATTT</entry></row></tbody></tgroup></table></tables>

0621Primers Used for Generating PCR Products to Serve as Substrates for T7 Transcription of sgRNAs for Gel-Based Deaminase Assay.

0622rev_gRNA_T7 was used in all cases. The pFYF1320 plasmid was used as template as noted in Materials and Methods section. SEQ ID NOs: 200-223 appear from top to bottom below, respectively.

0623<tables id="TABLE-US-00115" num="00115"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="133pt" align="left" /><colspec colname="2" colwidth="308pt" align="left" /><colspec colname="3" colwidth="0pt" align="left" /><tbody valign="top"><row><entry>rev_sgRNA_T7</entry><entry>AAAAAAAGCACCGACTCGGTG</entry><entry /></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_2</entry><entry>TAATACGACTCACTATAGGCCGCGGATTTATTTATTTAAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_3</entry><entry>TAATACGACTCACTATAGGTCCGCGGATTTATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_4</entry><entry>TAATACGACTCACTATAGGTTCCGCGGATTTATTTATTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_5</entry><entry>TAATACGACTCACTATAGGATTCCGCGGATTTATTTATTGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_6</entry><entry>TAATACGACTCACTATAGGTATTCCGCGGATTTATTTATGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_7</entry><entry>TAATACGACTCACTATAGGTTATTCCGCGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_8</entry><entry>TAATACGACTCACTATAGGATTATTCCGCGGATTTATTTGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_9</entry><entry>TAATACGACTCACTATAGGTATTATTCCGCGGATTTATTGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_10</entry><entry>TAATACGACTCACTATAGGATTATTATCCGCGGATTTATGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_11</entry><entry>TAATACGACTCACTATAGGTATTATATTCCGCGGATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>twd_sgRNA_T7_dsDNA_12</entry><entry>TAATACGACTCACTATAGGTTATTATATTCCGCGGATTTGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_13</entry><entry>TAATACGACTCACTATAGGATTATTATATTCCGCGGATTGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_14</entry><entry>TAATACGACTCACTATAGGTATTATTATATTCCGCGGATGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_15</entry><entry>TAATACGACTCACTATAGGATTATTATTATTACCGCGGAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_18</entry><entry>TAATACGACTCACTATAGGATTATTATTATTATTACCGCGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_noC</entry><entry>TAATACGACTCACTATAGGATATTAATTTATTTATTTAAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_APOE4_C112R</entry><entry>TAATACGACTCACTATAGGGGAGGACGTGCGCGGCCGCCGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_APOE4_C158R</entry><entry>TAATACGACTCACTATAGGGAAGCGCCTGGCAGTGTACCGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_CTNNB1_T41A</entry><entry>TAATACGACTCACTATAGGCTGTGGCAGTGGCACCAGAAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_HRAS_Q61R</entry><entry>TAATACGACTCACTATAGGCCTCCCGGCCGGCGGTATCCGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_53_Y163C</entry><entry>TAATACGACTCACTATAGGGCTTGCAGATGGCCATGGCGGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_53_Y236C</entry><entry>TAATACGACTCACTATAGGACACATGCAGTTGTAGTGGAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_dsDNA_53_N239D</entry><entry>TAATACGACTCACTATAGGTGTCACACATGTAGTTGTAGGTTTTAGAGCTAGAAATAGCA</entry></row></tbody></tgroup></table></tables>

0624Sequences of 80-Nucleotide Unlabeled Strands and Cy3-Labeled Universal Primer Used in Gel-Based dsDNA Deaminase Assays.

0625SEQ ID NOs: 224-248 appear from top to bottom below, respectively.

0626<tables id="TABLE-US-00116" num="00116"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="84pt" align="left" /><colspec colname="2" colwidth="357pt" align="left" /><colspec colname="3" colwidth="0pt" align="left" /><tbody valign="top"><row><entry>Cy3-primer</entry><entry>Cy3-GTAGGTAGTTAGGATGAATGGAAGGTTGGTA</entry><entry /></row><row><entry></entry></row><row><entry>dsDNA_2</entry><entry>GTCCATGGATCCAGAGGTCATCCATTAAATAAATAAATCCGCGGGGCTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_3</entry><entry>GTCCATGGATCCAGAGGTCATCCATAAATAAATAAATCCGCGGAAGCTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_4</entry><entry>GTCCATGGATCCAGAGGTCATCCATAATAAATAAATCCGCGGAAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_5</entry><entry>GTCCATGGATCCAGAGGTCATCCAAATAAATAAATCCGCGGAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_6</entry><entry>GTCCATGGATCCAGAGGTCATCCAATAAATAAATCCGCGGAATAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_7</entry><entry>GTCCATGGATCCAGAGGTCATCCATAAATAAATCCGCGGAATAAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_8</entry><entry>GTCCATGGATCCAGAGGTCATCCAAAATAAATCCGCGGAATAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_9</entry><entry>GTCCATGGATCCAGAGGTCATCCAAATAAATCCGCGGAATAATAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_10</entry><entry>GTCCATGGATCCAGAGGTCATCCAATAAATCCGCGGATAATAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_11</entry><entry>GTCCATGGATCCAGAGGTCATCCATAAATCCGCGGAATATAATAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_12</entry><entry>GTCCATGGATCCAGAGGTCATCCAAAATCCGCGGAATATAATAAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_13</entry><entry>GTCCATGGATCCAGAGGTCATCCAAATCCGCGGAATATAATAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_14</entry><entry>GTCCATGGATCCAGAGGTCATCCAATCCGCGGAATATAATAATAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_15</entry><entry>GTCCATGGATCCAGAGGTCATCCATCCGCGGTAATAATAATAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_18</entry><entry>GTCCATGGATCCAGAGGTCATCCAGCGGTAATAATAATAATAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_noC</entry><entry>GTCCATGGATCCAGAGGTCATCCATTAAATAAATAAATTAATATTACTATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_8U</entry><entry>5Cy3-GTAGGTAGTTAGGATGAATGGAAGGTTGGTGTAGATTATTATCUGCGGATTTATTGGATGACCTCTGGATCCATGGACAT</entry></row><row><entry></entry></row><row><entry>dsDNA_APOE_C112R</entry><entry>GCACCTCGCCGCGGTACTGCACCAGGCGGCCGCGCACGTCCTCCATGTCTACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_APOE_C158R</entry><entry>CGGCGCCCTCGCGGGCCCCGGCCTGGTACACTGCCAGGCGCTTCTGCAGTACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_CTNNB1_T41A</entry><entry>GTCTTACCTGGACTCTGGAATCCATTCTGGTGCCACTGCCACAGCTCCTTACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_HRAS_Q61R</entry><entry>GGAGACGTGCCTGTTGGACATCCTGGATACCGCCGGCCGGGAGGAGTACTACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_p53_Y163C</entry><entry>ACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTGCAAGCAGTCATACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_p53_Y236C</entry><entry>AGGTTGGCTCTGACTGTACCACCATCCACTACAACTGCATGTGTAACAGTACCAACCTTCCATTCATCCTAACTACCTAC</entry></row><row><entry></entry></row><row><entry>dsDNA_p53_N239D</entry><entry>TGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTGACAGTTCCTACCAACCTTCCATTCATCCTAACTACCTAC</entry></row></tbody></tgroup></table></tables>

0627Primers Used for Generating PCR Products to Serve as Substrates for T7 Transcription of sgRNAs for High-Throughput Sequencing.

0628rev_gRNA_T7 (above) was used in all cases. The pFYF1320 plasmid was used as template as noted in Materials and Methods section. SEQ ID NOs: 249-300 appear from top to bottom below, respectively.

0629<tables id="TABLE-US-00117" num="00117"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="133pt" align="left" /><colspec colname="2" colwidth="308pt" align="left" /><colspec colname="3" colwidth="0pt" align="left" /><tbody valign="top"><row><entry>fwd_sgRNA_T7_HTS_base</entry><entry>TAATACGACTCACTATAGGTTATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry><entry /></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_1A</entry><entry>TAATACGACTCACTATAGGATATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_1C</entry><entry>TAATACGACTCACTATAGGCTATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_1G</entry><entry>TAATACGACTCACTATAGGGTATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>Fwd_sgRNA_T7_HTS_2A</entry><entry>TAATACGACTCACTATAGGTAATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_2C</entry><entry>TAATACGACTCACTATAGGTCATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_2G</entry><entry>TAATACGACTCACTATAGGTGATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_3T</entry><entry>TAATACGACTCACTATAGGTTTTTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_3C</entry><entry>TAATACGACTCACTATAGGTTCTTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_3G</entry><entry>TAATACGACTCACTATAGGTTGTTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_4A</entry><entry>TAATACGACTCACTATAGGTTAATTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_4C</entry><entry>TAATACGACTCACTATAGGTTACTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_4G</entry><entry>TAATACGACTCACTATAGGTTAGTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_5A</entry><entry>TAATACGACTCACTATAGGTTATATCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_5C</entry><entry>TAATACGACTCACTATAGGTTATCTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_5G</entry><entry>TAATACGACTCACTATAGGTTATGTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_6A</entry><entry>TAATACGACTCACTATAGGTTATTACGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_6C</entry><entry>TAATACGACTCACTATAGGTTATTCCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_6G</entry><entry>TAATACGACTCACTATAGGTTATTGCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_8A</entry><entry>TAATACGACTCACTATAGGTTATTTCATGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_8T</entry><entry>TAATACGACTCACTATAGGTTATTTCTTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_8C</entry><entry>TAATACGACTCACTATAGGTTATTTCCTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_9A</entry><entry>TAATACGACTCACTATAGGTTATTTCGAGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_9C</entry><entry>TAATACGACTCACTATAGGTTATTTCGCGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_9G</entry><entry>TAATACGACTCACTATAGGTTATTTCGGGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_10A</entry><entry>TAATACGACTCACTATAGGTTATTTCGTAGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_10T</entry><entry>TAATACGACTCACTATAGGTTATTTCGTTGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_10C</entry><entry>TAATACGACTCACTATAGGTTATTTCGTCGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_11A</entry><entry>TAATACGACTCACTATAGGTTATTTCGTGAATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_11T</entry><entry>TAATACGACTCACTATAGGTTATTTCGTGTATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_11C</entry><entry>TAATACGACTCACTATAGGTTATTTCGTGCATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_12T</entry><entry>TAATACGACTCACTATAGGTTATTTCGTGGTTTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_12C</entry><entry>TAATACGACTCACTATAGGTTATTTCGTGGCTTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_12G</entry><entry>TAATACGACTCACTATAGGTTATTTCGTGGGTTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_13A</entry><entry>TAATACGACTCACTATAGGTTATTTCGTGGAATTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_13C</entry><entry>TAATACGACTCACTATAGGTTATTTCGTGGACTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_13G</entry><entry>TAATACGACTCACTATAGGTTATTTCGTGGAGTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_muitiC</entry><entry>TAATACGACTCACTATAGGTTCCCCCCCCGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_TCGCACCC_odd</entry><entry>TAATACGACTCACTATAGGCGCACCCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_CCTCGCAC_odd</entry><entry>TAATACGACTCACTATAGGCTCGCACGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_ACCCTCGC_odd</entry><entry>TAATACGACTCACTATAGGCCCTCGCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_GCACCCTC_odd</entry><entry>TAATACGACTCACTATAGGCACCCTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_TCGCACCC_even</entry><entry>TAATACGACTCACTATAGGTCGCACCCGTGGATTTATTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_CCTCGCAC_even</entry><entry>TAATACGACTCACTATAGGCCTCGCACGTGGATTTATTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_ACCCTCGC_even</entry><entry>TAATACGACTCACTATAGGACCCTCGCGTGGATTTATTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_GCACCCTC_even</entry><entry>TAATACGACTCACTATAGGGCACCCTCGTGGATTTATTAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_EMX1</entry><entry>TAATACGACTCACTATAGGGAGTCCGAGCAGAAGAAGAAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_FANCF</entry><entry>TAATACGACTCACTATAGGGGAATCCCTTCTGCAGCACCGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_HEK293_site2</entry><entry>TAATACGACTCACTATAGGGAACACAAAGCATAGACTGCGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_HEK293_site3</entry><entry>TAATACGACTCACTATAGGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_HEK293_site4</entry><entry>TAATACGACTCACTATAGGGGCACTGCGGCTGGAGGTGGGTTTTAGAGCTAGAAATAGCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HTS_RNF2</entry><entry>TAATACGACTCACTATAGGGTCATCTTAGTCATTACCTGGTTTTAGAGCTAGAAATAGCA</entry></row></tbody></tgroup></table></tables>

0630Sequences of In Vitro-Edited dsDNA for High-Throughput Sequencing (HTS).

0631Shown are the sequences of edited strands. Reverse complements of all sequences shown were also obtained. dsDNA substrates were obtained by annealing complementary strands as described in Materials and Methods. Oligonucleotides representing the EMX1, FANCF, HEK293 site 2, HEK293 site 3, HEK293 site 4, and RNF2 loci were originally designed for use in the gel-based deaminase assay and therefore have the same 25-nt sequence on their 5′-ends (matching that of the Cy3-primer). SEQ ID NOs: 301-352 appear from top to bottom below, respectively.

0632<tables id="TABLE-US-00118" num="00118"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="98pt" align="left" /><colspec colname="2" colwidth="343pt" align="left" /><colspec colname="3" colwidth="0pt" align="left" /><tbody valign="top"><row><entry>Base sequence</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry><entry /></row><row><entry></entry></row><row><entry>1A</entry><entry>ACGTAAACGGCCACAAGTTCATATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>1C</entry><entry>ACGTAAACGGCCACAAGTTCCTATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>1G</entry><entry>ACGTAAACGGCCACAAGTTCGTATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>2A</entry><entry>ACGTAAACGGCCACAAGTTCTAATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>2C</entry><entry>ACGTAAACGGCCACAAGTTCTCATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>2G</entry><entry>ACGTAAACGGCCACAAGTTCTGATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>3T</entry><entry>ACGTAAACGGCCACAAGTTCTTTTTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>3C</entry><entry>ACGTAAACGGCCACAAGTTCTTCTTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>3G</entry><entry>ACGTAAACGGCCACAAGTTCTTGTTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>4A</entry><entry>ACGTAAACGGCCACAAGTTCTTAATTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>4C</entry><entry>ACGTAAACGGCCACAAGTTCTTACTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>4G</entry><entry>ACGTAAACGGCCACAAGTTCTTAGTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>5A</entry><entry>ACGTAAACGGCCACAAGTTCTTATATCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>5C</entry><entry>ACGTAAACGGCCACAAGTTCTTATCTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>5G</entry><entry>ACGTAAACGGCCACAAGTTCTTATGTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>6A</entry><entry>ACGTAAACGGCCACAAGTTCTTATTACGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>6C</entry><entry>ACGTAAACGGCCACAAGTTCTTATTCCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>6G</entry><entry>ACGTAAACGGCCACAAGTTCTTATTGCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>8A</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCATGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>8T</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCTTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>8C</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCCTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>9A</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGAGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>9C</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGCGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>9G</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGGGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>10A</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGTAGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>10T</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGTTGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>10C</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGTCGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>11A</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGTGAATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>11T</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGTGTATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>11C</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGTGCATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>12T</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGTGGTTTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>12C</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGTGGCTTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>12G</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGTGGGTTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>13A</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGTGGAATTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>13C</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGTGGACTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>13G</entry><entry>ACGTAAACGGCCACAAGTTCTTATTTCGTGGAGTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>multiC</entry><entry>ACGTAAACGGCCACAAGTTCTTCCCCCCCCGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>TCGCACCC_odd</entry><entry>ACGTAAACGGCCACAAGTTTCGCACCCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>CCTCGCAC_odd</entry><entry>ACGTAAACGGCCACAAGTTCCTCGCACGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>ACCCTCGC_odd</entry><entry>ACGTAAACGGCCACAAGTTACCCTCGCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>GCACCCTC_odd</entry><entry>ACGTAAACGGCCACAAGTTGCACCCTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>TCGCACCC_even</entry><entry>ACGTAAACGGCCACAAGTATTCGCACCCGTGGATTTATTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>CCTCGCAC_even</entry><entry>ACGTAAACGGCCACAAGTATCCTCGCACGTGGATTTATTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>ACCCTCGC_even</entry><entry>ACGTAAACGGCCACAAGTATACCCTCGCGTGGATTTATTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>GCACCCTC_even</entry><entry>ACGTAAACGGCCACAAGTATGCACCCTCGTGGATTTATTATGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>EMX1_invitro</entry><entry>GTAGGTAGTTAGGATGAATGGAAGGTTGGTAGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTG</entry></row><row><entry></entry></row><row><entry>FANCF_invitro</entry><entry>GTAGGTAGTTAGGATGAATGGAAGGTTGGTACTCATGGAATCCCTTCTGCAGCACCTGGATCGCTTTTCCGAGCTTCTGG</entry></row><row><entry></entry></row><row><entry>HEK293_site2_invitro</entry><entry>GTAGGTAGTTAGGATGAATGGAAGGTTGGTAAACTGGAACACAAAGCATAGACTGCGGGGCGGGCCAGCCTGAATAGCTG</entry></row><row><entry></entry></row><row><entry>HEK293_site3_invitro</entry><entry>GTAGGTAGTTAGGATGAATGGAAGGTTGGTACTTGGGGCCCAGACTGAGCACGTGATGGCAGAGGAAAGGAAGCCCTGCT</entry></row><row><entry></entry></row><row><entry>HEK293_site4_invitro</entry><entry>GTAGGTAGTTAGGATGAATGGAAGGTTGGTACCGGTGGCACTGCGGCTGGAGGTGGGGGTTTAAGCGGAGACTCTGGTGC</entry></row><row><entry></entry></row><row><entry>RNF2_invitro</entry><entry>GTAGGTAGTTAGGATGAATGGAAGGTTGGTATGGCAGTCATCTTAGTCATTACCTGAGGTGTTCGTTGTAACTCATATAA</entry></row></tbody></tgroup></table></tables>

0633Primers for HTS of In Vitro Edited dsDNA.

0634SEQ ID NOs: 353-361 appear from top to bottom below, respectively.

0635<tables id="TABLE-US-00119" num="00119"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="105pt" align="left" /><colspec colname="2" colwidth="336pt" align="left" /><colspec colname="3" colwidth="0pt" align="left" /><tbody valign="top"><row><entry>fwd_invitro_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNACGTAAACGGCCACAA</entry><entry /></row><row><entry></entry></row><row><entry>rev_invitro_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCGTCCTTGAAGAAGATGC</entry></row><row><entry></entry></row><row><entry>fwd_invitro_HEK_targets</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTAGGTAGTTAGGATGAATGGAA</entry></row><row><entry></entry></row><row><entry>rev_EMX1_invitro</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCACCGGTTGATGTGATGG</entry></row><row><entry></entry></row><row><entry>rev_FANCF_invitro</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCCAGAAGCTCGGAAAAGC</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site2_invitro</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCAGCTATTCAGGCTGGC</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site3_invitro</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTAGCAGGGCTTCCTTTC</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site4_invitro</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGCACCAGAGTCTCCG</entry></row><row><entry></entry></row><row><entry>rev_RNF2_invitro</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTTATATGAGTTACAACGAACACC</entry></row></tbody></tgroup></table></tables>

0636Primers for HTS of On-Target and Off-Target Sites from all Mammalian Cell Culture Experiments.

0637SEQ ID NOs: 362-469 appear from top to bottom below, respectively.

0638<tables id="TABLE-US-00120" num="00120"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="112pt" align="left" /><colspec colname="2" colwidth="329pt" align="left" /><colspec colname="3" colwidth="0pt" align="left" /><tbody valign="top"><row><entry>fwd_EMX1_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCAGCTCAGCCTGAGTGTTGA</entry><entry /></row><row><entry></entry></row><row><entry>rev_EMX1_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCTCGTGGGTTTGTGGTTGC</entry></row><row><entry></entry></row><row><entry>fwd_FANCF_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCATTGCAGAGAGGCGTATCA</entry></row><row><entry></entry></row><row><entry>rev_FANCF_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGGGGTCCCAGGTGCTGAC</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site2_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCAGCCCCATCTGTCAAACT</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site2_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTGAATGGATTCCTTGGAAACAATGA</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site3_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNATGTGGGCTGCCTAGAAAGG</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site3_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCCCAGCCAAACTTGTCAACC</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site4_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGAACCCAGGTAGCCAGAGAC</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site4_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTTTCAACCCGAACGGAG</entry></row><row><entry></entry></row><row><entry>fwd_RNF2_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTCTTCTTTATTTCCAGCAATGT</entry></row><row><entry></entry></row><row><entry>rev_RNF2_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGTTTTCATGTTCTAAAAATGTATCCCA</entry></row><row><entry></entry></row><row><entry>fwd_p53_Y163C_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTACAGTACTCCCCTGCCCTC</entry></row><row><entry></entry></row><row><entry>rev_p53_Y163C_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGCTGCTCACCATCGCTATCT</entry></row><row><entry></entry></row><row><entry>fwd_p53_N239D_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCTCATCTTGGGCCTGTGTT</entry></row><row><entry></entry></row><row><entry>rev_p53_N239D_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTAAATCGGTAAGAGGTGGGCC</entry></row><row><entry></entry></row><row><entry>fwd_APOE4_C158R_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCGGACATGGAGGACGTG</entry></row><row><entry></entry></row><row><entry>rev_APOE4_C158R_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGTTCCACCAGGGGCCC</entry></row><row><entry></entry></row><row><entry>fwd_EMX1_off1_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCCAATCATTGATGCTTTT</entry></row><row><entry></entry></row><row><entry>rev_EMX1_off1_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTAGAAACATTTACCATAGACTATCACCT</entry></row><row><entry></entry></row><row><entry>fwd_EMX1_off2_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAGTAGCCTCTTTCTCAATGTGC</entry></row><row><entry></entry></row><row><entry>rev_EMX1_off2_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGCTTTCACAAGGATGCAGTCT</entry></row><row><entry></entry></row><row><entry>fwd_EMX1_off3_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGAGCTAGACTCCGAGGGGA</entry></row><row><entry></entry></row><row><entry>rev_EMX1_off3_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTCGTCCTGCTCTCACTT</entry></row><row><entry></entry></row><row><entry>fwd_EMX1_off4_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAGAGGCTGAAGAGGAAGACCA</entry></row><row><entry></entry></row><row><entry>rev_EMX1_off4_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGGCCCAGCTGTGCATTCTAT</entry></row><row><entry></entry></row><row><entry>fwd_EMX1_off6_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCAAGAGGGCCAAGTCCTG</entry></row><row><entry></entry></row><row><entry>rev_EMX1_off6_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCAGCGAGGAGTGACAGCC</entry></row><row><entry></entry></row><row><entry>fwd_EMX1_off7_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCACTCCACCTGATCTCGGGG</entry></row><row><entry></entry></row><row><entry>rev_EMX1_off7_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCGAGGAGGGAGGGAGCAG</entry></row><row><entry></entry></row><row><entry>fwd_EMX1_off8_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNACCACAAATGCCCAAGAGAC</entry></row><row><entry></entry></row><row><entry>rev_EMX1_off8_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGACACAGTCAAGGGCCGG</entry></row><row><entry></entry></row><row><entry>fwd_EMX1_off9_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCCACCTTTGAGGAGGCAAA</entry></row><row><entry></entry></row><row><entry>rev_EMX1_off9_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTTCCATCTGAGAAGAGAGTGGT</entry></row><row><entry></entry></row><row><entry>fwd_EMX1_off10_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTCATACCTTGGCCCTTCCT</entry></row><row><entry></entry></row><row><entry>rev_EMX1_off10_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTCCCTAGGCCCACACCAG</entry></row><row><entry></entry></row><row><entry>fwd_FANCF_off1_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAACCCACTGAAGAAGCAGGG</entry></row><row><entry></entry></row><row><entry>rev_FANCF_off1_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGGTGCTTAATCCGGCTCCAT</entry></row><row><entry></entry></row><row><entry>fwd_FANCF_off2_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCAGTGTTTCCATCCCGAA</entry></row><row><entry></entry></row><row><entry>rev_FANCF_off2_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCCTCTGACCTCCACAACTCT</entry></row><row><entry></entry></row><row><entry>fwd_FANCF_off3_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTNNNNCTGGGTACAGTTCTGCGTGT</entry></row><row><entry></entry></row><row><entry>rev_FANCF_off3_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTCACTCTGAGCATCGCCAAG</entry></row><row><entry></entry></row><row><entry>fwd_FANCF_off4_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGTTTAGAGCCAGTGAACTAGAG</entry></row><row><entry></entry></row><row><entry>rev_FANCF_off4_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGCAAGACAAAATCCTCTTTATACTTTG</entry></row><row><entry></entry></row><row><entry>fwd_FANCF_off5_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGGAGGGGACGGCCTTAC</entry></row><row><entry></entry></row><row><entry>rev_FANCF_off5_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGCCTCTGGCGAACATGGC</entry></row><row><entry></entry></row><row><entry>fwd_FANCF_off6_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCTGGTTAAGAGCATGGGC</entry></row><row><entry></entry></row><row><entry>rev_FANCF_off6_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGATTGAGTCCCCACAGCACA</entry></row><row><entry></entry></row><row><entry>fwd_FANCF_off7_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCAGTGTTTCCCATCCCCAA</entry></row><row><entry></entry></row><row><entry>rev_FANCF_off7_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTGACCTCCACAACTGGAAAAT</entry></row><row><entry></entry></row><row><entry>fwd_FANCF_off8_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCTTCCAGACCCACCTGAAG</entry></row><row><entry></entry></row><row><entry>rev_FANCF_off8_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTACCGAGGAAAATTGCTTGTCG</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site2_off1_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTGTGGAGAGTGAGTAAGCCA</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site2_off1_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTACGGTAGGATGATTTCAGGCA</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site2_off2_HTS</entry><entry>ACACTCTTTCCCTACACGACgCTCTTCCGATCTNNNNCACAAAGCAGTGTAGCTCAGG</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site2_off2_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTTTTTGGTACTCGAGTGTTATTCAG</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site3_off1_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCCCTGTTGACCTGGAGAA</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site3_off1_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCACTGTACTTGCCCTGACCA</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site3_off2_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTGGTGTTGACAGGGAGCAA</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site3_off2_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGAGATGTGGGCAGAAGGG</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site3_off3_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGAGAGGGAACAGAAGGGCT</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site3_off3_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCCAAAGGCCCAAGAACCT</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site3_off4_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCTAGCACTTTGGAAGGTCG</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site3_off4_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGCTCATCTTAATCTGCTCAGCC</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site3_off5_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAAAGGAGCAGCTCTTCCTGG</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site3_off5_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTGCACCATCTCCCACAA</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site4_off1_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGCATGGCTTCTGAGACTCA</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site4_off1_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTCCCTTGCACTCCCTGTCTTT</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site4_off2_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTTGGCAATGGAGGCATTGG</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site4_off2_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGAAGAGGCTGCCCATGAGAG</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site4_off3_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGTCTGAGGCTCGAATCCTG</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site4_off3_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGTGGCCTCCATATCCCTG</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site4_off4_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTTCCACCAGAACTCAGCCC</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site4_off4_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCCTCGGTTCCTCCACAACAC</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site4_off5_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCACGGGAAGGACAGGAGAAC</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site4_off5_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGCAGGGGAGGGATAAAGCAG</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site4_off6_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCACGGGAGATGGCTTATGT</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site4_off6_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCACATCCTCACTGTGCCACT</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site4_off7_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTCAGTCTCGGCCCCTCA</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site4_off7_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGCCACTGTAAAGCTCTTGGG</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site4_off8_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAGGGTAGAGGGACAGAGCTG</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site4_off8_HTS</entry><entry>TGgAGTTCAGACGTGTGCTCTTCCGATCTGGACCCCACATAGTCAGTGC</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site4_off9_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCTGTCAGCCCTATCTCCATC</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site4_off9_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTGGGCAATTAGGACAGGGAC</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site4_off10_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCAGCGGAGGAGGTAGATTG</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site4_off10_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCTCAGTACCTGGAGTCCCGA</entry></row><row><entry></entry></row><row><entry>fwd_HEK2_ChIP_off1_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGACAGGCTCAGgAAAGCTGT</entry></row><row><entry></entry></row><row><entry>rev_HEK2_ChIP_off1_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTACACAAGCCTTTCTCCAGGG</entry></row><row><entry></entry></row><row><entry>fwd_HEK2_ChIP_off2_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAATAGGGGGTGAGACTGGGG</entry></row><row><entry></entry></row><row><entry>rev_HEK2_ChIP_off2_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGCCTCAGACGAGACTTGAGG</entry></row><row><entry></entry></row><row><entry>fwd_HEK2_ChIP_off3_HTS</entry><entry>ACAGTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGCCAGCAGGAAAGGAATCT</entry></row><row><entry></entry></row><row><entry>rev HEK2 ChIP off3 HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTGACTGCACCTGTAGCCATG</entry></row><row><entry></entry></row><row><entry>fwd_HEK2_ChIP_off4_HTS</entry><entry>ACACTCTTTCCCTAGACGACGCTCTTCCGATCTNNNNTCAAGGAAATCACCCTGCCC</entry></row><row><entry></entry></row><row><entry>rev_HEK2_ChIP_off4_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTAACTTCCTTGGTGTGCAGCT</entry></row><row><entry></entry></row><row><entry>fwd_HEK2_ChIP_off5_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNATGGGCTCAGCTACGTCATG</entry></row><row><entry></entry></row><row><entry>rev HEK2 ChIP off5 HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTAATAGCAGTGTGGTGGGCAA</entry></row><row><entry></entry></row><row><entry>fwd_HEK3_ChIP_off1_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCGCACATCCCTTGTCTCTCT</entry></row><row><entry></entry></row><row><entry>rev_HEK3_ChIP_off1_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCTACTGGAGCACACCCCAAG</entry></row><row><entry></entry></row><row><entry>fwd_HEK3_ChIP_off2_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGGGTCACGTAGCTTTGGTC</entry></row><row><entry></entry></row><row><entry>rev_HEK3_ChIP_off2_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTGGTGGCCATGTGCAACTAA</entry></row><row><entry></entry></row><row><entry>fwd_HEK3_ChIP_off3_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTACTACGTGCCAGCTCAGG</entry></row><row><entry></entry></row><row><entry>rev_HEK3_ChIP_off3_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTACCTCCCCTCCTCACTAACC</entry></row><row><entry></entry></row><row><entry>fwd_HEK3_ChIP_off4_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCCTCAGCTCCATTTCCTGT</entry></row><row><entry></entry></row><row><entry>rev_HEK3_ChIP_off4_HTS</entry><entry>TGAGTTCAGACGTGTGCTCTTCCGATCTAACCTTTATGGCACCAGGGG</entry></row><row><entry></entry></row><row><entry>fwd_HEK3_ChIP_off5_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGAGCTCAGCATTAGCAGGCT</entry></row><row><entry></entry></row><row><entry>rev_HEK3_ChIP_off5_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTTCCTGGCTTTCCGATTCCC</entry></row><row><entry></entry></row><row><entry>fwd_HEK4_ChIP_off1_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTGCAATTGGAGGAGGAGCT</entry></row><row><entry></entry></row><row><entry>rev_HEK4_ChIP_off1_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCACCAGCTACAGGCAGAACA</entry></row><row><entry></entry></row><row><entry>fwd_HEK4_ChIP_off3_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCTACCCCCAACACAGATGG</entry></row><row><entry></entry></row><row><entry>rev_HEK4_ChIP_off3_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCCACACAACTCAGGTCCTCC</entry></row></tbody></tgroup></table></tables>

0639Sequences of Single-Stranded Oligonucleotide Donor Templates (ssODNs) Used in HDR Studies.

0640<tables id="TABLE-US-00121" num="00121"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="308pt" align="left" /><colspec colname="2" colwidth="133pt" align="left" /><tbody valign="top"><row><entry>EMX1 sense</entry><entry /></row><row><entry>(SEQ ID NO: 470)</entry><entry /></row><row><entry>TCATCTGTGCCCCTCCCTCCCTGGCCCAGGTGAAGGTGTGGTTCCAGAACCGGAGGACAAAGTACA</entry><entry /></row><row><entry></entry></row><row><entry>AACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTTTGAGCAGAAGAAGAAGGGCTCCCATCACATC</entry></row><row><entry></entry></row><row><entry>AACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACCTCCAATGACTAG</entry></row><row><entry></entry></row><row><entry>GGT</entry></row><row><entry></entry></row><row><entry>EMX1 antisense</entry></row><row><entry>(SEQ ID NO: 471)</entry><entry /></row><row><entry>ACCCTAGTCATTGGAGGTGACATCGATGTCCTCCCCATTGGCCTGCTTCGTGGCAATGCGCCACCG</entry><entry /></row><row><entry></entry></row><row><entry>GTTGATGTGATGGGAGCCCTTCTTCTTCTGCTCAAACTGAGGCCCTTCCTCCTCCAGCTTCTGCCGT</entry></row><row><entry></entry></row><row><entry>TTGTACTTTGTCCTCCGGTTCTGGAACCACACCTTCACCTGGGCCAGGGAGGGAGGGGCACAGATGA</entry></row><row><entry></entry></row><row><entry>HEK293 site 3 sense</entry></row><row><entry>(SEQ ID NO: 472)</entry><entry /></row><row><entry>CATGCAATTAGTCTATTTCTGCTGCAAGTAAGCATGCATTTGTAGGCTTGATGCTTTTTTTCTGCTTCT</entry><entry /></row><row><entry></entry></row><row><entry>CCAGCCCTGGCCTGGGTCAATCCTTGGGGCTTAGACTGAGCACGTGATGGCAGAGGAAAGGAAGC</entry></row><row><entry></entry></row><row><entry>CCTGCTTCCTCCAGAGGGCGTCGCAGGACAGCTTTTCCTAGACAGGGGCTAGTATGTGCAGCTCCT</entry></row><row><entry></entry></row><row><entry>HEK293 site 3 antisense</entry></row><row><entry>(SEQ ID NO: 473)</entry><entry /></row><row><entry>AGGAGCTGCACATACTAGCCCCTGTCTAGGAAAAGCTGTCCTGCGACGCCCTCTGGAGGAAGCAGG</entry><entry /></row><row><entry></entry></row><row><entry>GCTTCCTTTCCTCTGCCATCACGTGCTCAGTCTAAGCCCCAAGGATTGACCCAGGCCAGGGCTGGA</entry></row><row><entry></entry></row><row><entry>GAAGCAGAAAAAAAGCATCAAGCCTACAAATGCATGCTTACTTGCAGCAGAAATAGACTAATTGCATG</entry></row><row><entry></entry></row><row><entry>HEK site 4 sense</entry></row><row><entry>(SEQ ID NO: 474)</entry><entry /></row><row><entry>GGCTGACAAAGGCCGGGCTGGGTGGAAGGAAGGGAGGAAGGGCGAGGCAGAGGGTCCAAAGCAG</entry><entry /></row><row><entry></entry></row><row><entry>GATGACAGGCAGGGGCACCGCGGCGCCCCGGTGGCATTGCGGCTGGAGGTGGGGGTTAAAGCGG</entry></row><row><entry></entry></row><row><entry>AGACTCTGGTGCTGTGTGACTACAGTGGGGGCCCTGCCCTCTCTGAGCCCCCGCCTCCAGGCCTGT</entry></row><row><entry></entry></row><row><entry>GTGTGT</entry></row><row><entry></entry></row><row><entry>HEK site 4 antisense</entry></row><row><entry>(SEQ ID NO: 475)</entry><entry /></row><row><entry>ACACACACAGGCCTGGAGGCGGGGGCTCAGAGAGGGCAGGGCCCCCACTGTAGTCACACAGCACC</entry><entry /></row><row><entry></entry></row><row><entry>AGAGTCTCCGCTTTAACCCCCACCTCCAGCCGCAATGCCACCGGGGCGCCGCGGTGCCCCTGCCT</entry></row><row><entry></entry></row><row><entry>GTCATCCTGCTTTGGACCCTCTGCCTCGCCCTTCCTCCCTTCCTTCCACCCAGCCCGGCCTTTGTCA</entry></row><row><entry></entry></row><row><entry>GCC</entry></row><row><entry></entry></row><row><entry>APOE4 sense</entry></row><row><entry>(SEQ ID NO: 476)</entry><entry /></row><row><entry>AGCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGTAAGCGGCTCCTCCG</entry><entry /></row><row><entry></entry></row><row><entry>CGATGCCGATGACCTGCAGAAGTGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGAG</entry></row><row><entry></entry></row><row><entry>CGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGGGCCG</entry></row><row><entry></entry></row><row><entry>CCACTGT</entry></row><row><entry></entry></row><row><entry>APOE4 antisense</entry></row><row><entry>(SEQ ID NO: 477)</entry><entry /></row><row><entry>ACAGTGGCGGCCCGCACGCGGCCCTGTTCCACCAGGGGCCCCAGGCGCTCGCGGATGGCGCTGA</entry><entry /></row><row><entry></entry></row><row><entry>GGCCGCGCTCGGCGCCCTCGCGGGCCCCGGCCTGGTACACTGCCAGGCACTTCTGCAGGTCATCG</entry></row><row><entry></entry></row><row><entry>GCATCGCGGAGGAGCCGCTTACGCAGCTTGCGCAGGTGGGAGGCGAGGCGCACCCGCAGCTCCT</entry></row><row><entry></entry></row><row><entry>CGGTGCT</entry></row><row><entry></entry></row><row><entry>p53 Y163C sense</entry></row><row><entry>(SEQ ID NO: 478)</entry><entry /></row><row><entry>ACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGA</entry><entry /></row><row><entry></entry></row><row><entry>TTCCACACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGAC</entry></row><row><entry></entry></row><row><entry>GGAGGTTGTGAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTGAGCAGCTGGGGC</entry></row><row><entry></entry></row><row><entry>TG</entry></row><row><entry></entry></row><row><entry>p53 Y163C antisense</entry></row><row><entry>(SEQ ID NO: 479)</entry><entry /></row><row><entry>CAGCCCCAGCTGCTCACCATCGCTATCTGAGCAGCGCTCATGGTGGGGGCAGCGCCTCACAACCTC</entry><entry /></row><row><entry></entry></row><row><entry>CGTCATGTGCTGTGACTGCTTGTAGATGGCCATGGCGCGGACGCGGGTGCCGGGCGGGGGTGTGG</entry></row><row><entry></entry></row><row><entry>AATCAACCCACAGCTGCACAGGGCAGGTCTTGGCCAGTTGGCAAAACATCTTGTTGAGGGCAGGGG</entry></row><row><entry></entry></row><row><entry>AGT</entry></row></tbody></tgroup></table></tables>

0641Deaminase Gene gBlocks Gene Fragments

0642<tables id="TABLE-US-00122" num="00122"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="308pt" align="left" /><colspec colname="2" colwidth="133pt" align="left" /><tbody valign="top"><row><entry>hAID</entry><entry /></row><row><entry>(SEQ ID NO: 169)</entry><entry /></row><row><entry>rAPOBEC1 (mammalian)</entry><entry /></row><row><entry>(SEQ ID NO: 170)</entry><entry /></row><row><entry>CATCCTTGGTACCGAGCTCGGATCCAGCCACCATGAGCTCAGAGACTGGCCCAGTGGCTGTGGACC</entry><entry /></row><row><entry></entry></row><row><entry>CCACATTGAGACGGCGGATCGAGCCCCATGAGTTTGAGGTATTCTTCGATCCGAGAGAGCTCCGCA</entry></row><row><entry></entry></row><row><entry>AGGAGACCTGCCTGCTTTACGAAATTAATTGGGGGGGCCGGCACTCCATTTGGCGACATACATCACA</entry></row><row><entry></entry></row><row><entry>GAACACTAACAAGCACGTCGAAGTCAACTTCATCGAGAAGTTCACGACAGAAAGATATTTCTGTCCG</entry></row><row><entry></entry></row><row><entry>AACACAAGGTGCAGCATTACCTGGTTTCTCAGCTGGAGCCCATGCGGCGAATGTAGTAGGGCCATC</entry></row><row><entry></entry></row><row><entry>ACTGAATTCCTGTCAAGGTATCCCCACGTCACTCTGTTTATTTACATCGCAAGGCTGTACCACCACGC</entry></row><row><entry></entry></row><row><entry>TGACCCCCGCAATCGACAAGGCCTGCGGGATTTGATCTCTTCAGGTGTGACTATCCAAATTATGACT</entry></row><row><entry></entry></row><row><entry>GAGCAGGAGTCAGGATACTGCTGGAGAAACTTGTGAATTATAGCCCGAGTAATGAAGCCCACTGG</entry></row><row><entry></entry></row><row><entry>CCTAGGTATCCCCATCTGTGGGTACGACTGTACGTTCTTGAACTGTACTGCATCATACTGGGCCTGC</entry></row><row><entry></entry></row><row><entry>CTCCTTGTCTCAACATTCTGAGAAGGAAGCAGCCACAGCTGACATTCTTTACCATCGCTCTTCAGTCT</entry></row><row><entry></entry></row><row><entry>TGTCATTACCAGCGACTGCCCCCACACATTCTCTGGGCCACCGGGTTGAAATGAGCGGCCGCTCGA</entry></row><row><entry></entry></row><row><entry>TTGGTTTGGTGTGGCTCTAA</entry></row><row><entry></entry></row><row><entry>pmCDA1</entry></row><row><entry>(SEQ ID NO: 171)</entry><entry /></row><row><entry>CATCCTTGGTACCGAGCTCGGATCCAGCCACCATGACAGACGCTGAATATGTTAGGATCCATGAAAA</entry><entry /></row><row><entry></entry></row><row><entry>ACTGGATATCTATACATTTAAGAAGCAGTTCTTCAATAACAAAAAGTCAGTATCTCACAGATGCTATGT</entry></row><row><entry></entry></row><row><entry>CCTGTTCGAACTCAAGAGAAGAGGAGAAAGGCGGGCCTGTTTCTGGGGGTACGCGGTTAATAAACC</entry></row><row><entry></entry></row><row><entry>CCAGTCCGGGACCGAGAGGGGGATTCACGCCGAGATCTTTTCAATTAGGAAGGTTGAAGAGTATCT</entry></row><row><entry></entry></row><row><entry>TCGCGACAATCCCGGTCAGTTCACAATTAACTGGTACAGCTCCTGGAGCCCTTGCGCTGATTGCGCC</entry></row><row><entry></entry></row><row><entry>GAGAAAATACTCGAATGGTACAACCAGGAGTTGAGAGGCAATGGCCACACTCTCAAGATTTGGGCTT</entry></row><row><entry></entry></row><row><entry>GCAAGCTTTACTACGAGAAGAACGCGAGAAATCAGATTGGCTTGTGGAACCTCAGGGACAACGGGG</entry></row><row><entry></entry></row><row><entry>TCGGGTTGAATGTTATGGTGTCCGAACATTACCAGTGCTGTAGAAAGATCTTCATTCAGTCCAGTCAC</entry></row><row><entry></entry></row><row><entry>AATCAGCTGAACGAGAACAGATGGCTGGAGAAAACACTGAAACGGGCAGAGAAAAGGCGCTCAGAG</entry></row><row><entry></entry></row><row><entry>CTGAGTATCATGATCCAGGTCAAAATCCTGCATACAACCAAAAGCCCGGCTGTATAAGCGGCCGCTC</entry></row><row><entry></entry></row><row><entry>GATTGGTTTGGTGTGGCTCTAA</entry></row><row><entry></entry></row><row><entry>haPOBEC3G</entry></row><row><entry>(SEQ ID NO: 172)</entry><entry /></row><row><entry>CATCCTTGGTACCGAGCTCGGATCCAGCCACCATGGAGCTGAAGTATCACCCTGAGATGCGGTTTTT</entry><entry /></row><row><entry></entry></row><row><entry>CCACTGGTTTAGTAAGTGGCGCAAACTTCATCGGGATCAGGAGTATGAAGTGACCTGGTATATCTCT</entry></row><row><entry></entry></row><row><entry>TGGTCTCCCTGCACAAAATGTACACGCGACATGGCCACATTTCTGGCCGAGGATCCAAAGGTGACG</entry></row><row><entry></entry></row><row><entry>CTCACAATCTTTGTGGCCCGCCTGTATTATTTCTGGGACCCGGATTATCAGGAGGCACTTAGGTCAT</entry></row><row><entry></entry></row><row><entry>TGTGCCAAAAGCGCGACGGACCACGGGCGACTATGAAAATCATGAATTATGACGAATTCCAGCATTG</entry></row><row><entry></entry></row><row><entry>CTGGAGTAAGTTTGTGTACAGCCAGCGGGAGCTGTTCGAGCCCTGGAACAATCTTCCCAAGTACTAC</entry></row><row><entry></entry></row><row><entry>ATACTGCTTCACATTATGTTGGGGGAGATCCTTCGGCACTCTATGGATCCTCCTACCTTTACGTTAA</entry></row><row><entry></entry></row><row><entry>CTTTAATAATGAGCCTTGGGTTCGCGGGCGCCATGAAACCTATTTGTGCTACGAGGTCGAGCGGATG</entry></row><row><entry></entry></row><row><entry>CATAATGATACGTGGGTCCTGCTGAATCAGAGGAGGGGGTTTCTGTGTAACCAGGCTCCACATAAAC</entry></row><row><entry></entry></row><row><entry>ATGGATTTCTCGAGGGGCGGCACGCCGAACTGTGTTTCCTTGATGTGATACCTTTCTGGAAGCTCGA</entry></row><row><entry></entry></row><row><entry>CCTTGATCAAGATTACAGGGTGACGTGTTTCACCTCCTGGTCACCCTGCTTCAGTTGCGCCCAAGAG</entry></row><row><entry></entry></row><row><entry>ATGGCTAAATTTATCAGTAAGAACAAGCATGTGTCCCTCTGTATTTTTACAGCCAGAATTTATGATGAC</entry></row><row><entry></entry></row><row><entry>CAGGGCCGGTGCCAGGAGGGGCTGCGGACACTCGCTGAGGCGGGCGCGAAGATCAGCATAATGA</entry></row><row><entry></entry></row><row><entry>CATACTCCGAATTCAAACACTGTTGGGACACTTTTGTGGACCACCAGGGCTGCCCATTTCAGCCGTG</entry></row><row><entry></entry></row><row><entry>GGATGGGCTCGACGAACATAGTCAGGATCTCTCAGGCCGGCTGCGAGCCATATTGCAGAACCAGGA</entry></row><row><entry></entry></row><row><entry>GAATTAGGCGGCCGCTCGATTGGTTTGGTGTGGCTCTAA</entry></row><row><entry></entry></row><row><entry>rAPOBEC1(<i>E. Coli</i>)</entry></row><row><entry>(SEQ ID NO: 173)</entry><entry /></row><row><entry>GGCCGGGGATTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGATGTCTTCTGAAA</entry><entry /></row><row><entry></entry></row><row><entry>CCGGTCCGGTTGCGGTTGACCCGACCCTGCGTCGTCGTATCGAACCGCACGAATTCGAAGTTTTCT</entry></row><row><entry></entry></row><row><entry>TCGACCCGCGTGAACTGCGTAAAGAAACCTGCCTGCTGTACGAAATCAACTGGGGTGGTCGTCACT</entry></row><row><entry></entry></row><row><entry>CTATCTGGCGTCACACCTCTCAGAACACCAACAAACACGTTGAAGTTAACTTCATCGAAAAATTCACC</entry></row><row><entry></entry></row><row><entry>ACCGAACGTTACTTCTGCCCGAACACCCGTTGCTCTATCACCTGGTTCCTGTCTTGGTCTCCGTGCG</entry></row><row><entry></entry></row><row><entry>GTGAATGCTCTCGTGCGATCACCGAATTCCTGTCTCGTTACCCGCACGTTACCCTGTTCATCTACATC</entry></row><row><entry></entry></row><row><entry>GCGCGTCTGTACCACCACGCGGACCCGCGTAACCGTCAGGGTCTGCGTGACCTGATCTCTTCTGGT</entry></row><row><entry></entry></row><row><entry>GTTACCATCCAGATCATGACCGAACAGGAATCTGGTTACTGCTGGCGTAACTTCGTTAACTACTCTCC</entry></row><row><entry></entry></row><row><entry>GTCTAACGAAGCGCACTGGCCGCGTTACCCGCACCTGTGGGTTCGTCTGTACGTTCTGGAACTGTA</entry></row><row><entry></entry></row><row><entry>CTGCATCATCCTGGGTCTGCCGCCGTGCCTGAACATCCTGCGTCGTAAACAGCCGCAGCTGACCTT</entry></row><row><entry></entry></row><row><entry>CTTCACCATCGCGCTGCAGTCTTGCCACTACCAGCGTCTGCCGCCGCACATCCTGTGGGCGACCGG</entry></row><row><entry></entry></row><row><entry>TCTGAAAGGTGGTAGTGGAGGGAGCGGCGGTTCAATGGATAAGAAATAC</entry></row></tbody></tgroup></table></tables>

0643Amino Acid Sequences of NBE1, NBE2, and NBE3.

0000NBE1 for <i>E. Coli </i>Expression (His₆-rAPOBEC1-XTEN-dCas9) (SEQ ID NO: 154)

0644<tables id="TABLE-US-00123" num="00123"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MGSSHHHHHHMSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLY</entry></row><row><entry></entry></row><row><entry>EINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW</entry></row><row><entry></entry></row><row><entry>SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVT</entry></row><row><entry></entry></row><row><entry>IQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLP</entry></row><row><entry></entry></row><row><entry>PCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSES</entry></row><row><entry></entry></row><row><entry>ATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN</entry></row><row><entry></entry></row><row><entry>LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDS</entry></row><row><entry></entry></row><row><entry>FFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDST</entry></row><row><entry></entry></row><row><entry>DKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF</entry></row><row><entry></entry></row><row><entry>EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL</entry></row><row><entry></entry></row><row><entry>GLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNL</entry></row><row><entry></entry></row><row><entry>SDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEK</entry></row><row><entry></entry></row><row><entry>YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE</entry></row><row><entry></entry></row><row><entry>DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTF</entry></row><row><entry></entry></row><row><entry>RIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT</entry></row><row><entry></entry></row><row><entry>NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKK</entry></row><row><entry></entry></row><row><entry>AIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHD</entry></row><row><entry></entry></row><row><entry>LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDK</entry></row><row><entry></entry></row><row><entry>VMKQLKRRRYTGWGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM</entry></row><row><entry></entry></row><row><entry>QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVV</entry></row><row><entry></entry></row><row><entry>DELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQ</entry></row><row><entry></entry></row><row><entry>ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQ</entry></row><row><entry></entry></row><row><entry>SFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ</entry></row><row><entry></entry></row><row><entry>RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD</entry></row><row><entry></entry></row><row><entry>ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVG</entry></row><row><entry></entry></row><row><entry>TALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN</entry></row><row><entry></entry></row><row><entry>FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNI</entry></row><row><entry></entry></row><row><entry>VKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVL</entry></row><row><entry></entry></row><row><entry>VVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDL</entry></row><row><entry></entry></row><row><entry>IIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEK</entry></row><row><entry></entry></row><row><entry>LKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAY</entry></row><row><entry></entry></row><row><entry>NKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD</entry></row><row><entry></entry></row><row><entry>ATLIHQSITGLYETRIDLSQLGGDSGGSPKKKRKV</entry></row></tbody></tgroup></table></tables><br /> NBE1 for Mammalian Expression (rAPOBEC1-XTEN-dCas9-NLS) (SEQ ID NO: 155)

0645<tables id="TABLE-US-00124" num="00124"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYS</entry></row><row><entry></entry></row><row><entry>IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG</entry></row><row><entry></entry></row><row><entry>ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL</entry></row><row><entry></entry></row><row><entry>VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL</entry></row><row><entry></entry></row><row><entry>ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV</entry></row><row><entry></entry></row><row><entry>DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF</entry></row><row><entry></entry></row><row><entry>DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL</entry></row><row><entry></entry></row><row><entry>RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK</entry></row><row><entry></entry></row><row><entry>NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD</entry></row><row><entry></entry></row><row><entry>NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA</entry></row><row><entry></entry></row><row><entry>RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK</entry></row><row><entry></entry></row><row><entry>VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN</entry></row><row><entry></entry></row><row><entry>RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF</entry></row><row><entry></entry></row><row><entry>LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY</entry></row><row><entry></entry></row><row><entry>TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE</entry></row><row><entry></entry></row><row><entry>DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP</entry></row><row><entry></entry></row><row><entry>ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL</entry></row><row><entry></entry></row><row><entry>QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKV</entry></row><row><entry></entry></row><row><entry>LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG</entry></row><row><entry></entry></row><row><entry>GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI</entry></row><row><entry></entry></row><row><entry>TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES</entry></row><row><entry></entry></row><row><entry>EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE</entry></row><row><entry></entry></row><row><entry>IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS</entry></row><row><entry></entry></row><row><entry>KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK</entry></row><row><entry></entry></row><row><entry>LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL</entry></row><row><entry></entry></row><row><entry>ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ</entry></row><row><entry></entry></row><row><entry>LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA</entry></row><row><entry></entry></row><row><entry>ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY</entry></row><row><entry></entry></row><row><entry>ETRIDLSQLGGDSGGSPKKKRKV</entry></row></tbody></tgroup></table></tables><br /> Alternative NBE1 for Mammalian Expression with Human APOBEC1 (hAPOBEC1-XTEN-dCas9-NLS) (SEQ ID NO: 158)

0646<tables id="TABLE-US-00125" num="00125"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKI</entry></row><row><entry></entry></row><row><entry>WRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAI</entry></row><row><entry></entry></row><row><entry>REFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYY</entry></row><row><entry></entry></row><row><entry>HCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQ</entry></row><row><entry></entry></row><row><entry>NHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWRGSETPGTSESATPE</entry></row><row><entry></entry></row><row><entry>SDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA</entry></row><row><entry></entry></row><row><entry>LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR</entry></row><row><entry></entry></row><row><entry>LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD</entry></row><row><entry></entry></row><row><entry>LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP</entry></row><row><entry></entry></row><row><entry>INASGVDAKAILSARLSKSRRLENLIAQLGEKKNGLFGNLIALSLGLTPN</entry></row><row><entry></entry></row><row><entry>FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL</entry></row><row><entry></entry></row><row><entry>LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF</entry></row><row><entry></entry></row><row><entry>FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK</entry></row><row><entry></entry></row><row><entry>QRTFDNGSPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV</entry></row><row><entry></entry></row><row><entry>GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNL</entry></row><row><entry></entry></row><row><entry>PNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL</entry></row><row><entry></entry></row><row><entry>FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK</entry></row><row><entry></entry></row><row><entry>DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK</entry></row><row><entry></entry></row><row><entry>RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL</entry></row><row><entry></entry></row><row><entry>TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMG</entry></row><row><entry></entry></row><row><entry>RHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE</entry></row><row><entry></entry></row><row><entry>NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSI</entry></row><row><entry></entry></row><row><entry>DNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK</entry></row><row><entry></entry></row><row><entry>AERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE</entry></row><row><entry></entry></row><row><entry>VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP</entry></row><row><entry></entry></row><row><entry>KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL</entry></row><row><entry></entry></row><row><entry>ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT</entry></row><row><entry></entry></row><row><entry>GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG</entry></row><row><entry></entry></row><row><entry>KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS</entry></row><row><entry></entry></row><row><entry>LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN</entry></row><row><entry></entry></row><row><entry>EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI</entry></row><row><entry></entry></row><row><entry>REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI</entry></row><row><entry></entry></row><row><entry>TGLYETRIDLSQLGGDSGGSPKKKRKV</entry></row></tbody></tgroup></table></tables><br /> NBE2 (rAPOBEC1-XTEN-dCas9-UGI-NLS) (SEQ ID NO: 156)

0647<tables id="TABLE-US-00126" num="00126"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYS</entry></row><row><entry></entry></row><row><entry>IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG</entry></row><row><entry></entry></row><row><entry>ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF</entry></row><row><entry></entry></row><row><entry>LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIY</entry></row><row><entry></entry></row><row><entry>LALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG</entry></row><row><entry></entry></row><row><entry>VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN</entry></row><row><entry></entry></row><row><entry>FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI</entry></row><row><entry></entry></row><row><entry>LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQS</entry></row><row><entry></entry></row><row><entry>KNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF</entry></row><row><entry></entry></row><row><entry>DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL</entry></row><row><entry></entry></row><row><entry>ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNE</entry></row><row><entry></entry></row><row><entry>KVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKT</entry></row><row><entry></entry></row><row><entry>NRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD</entry></row><row><entry></entry></row><row><entry>FLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRR</entry></row><row><entry></entry></row><row><entry>YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFK</entry></row><row><entry></entry></row><row><entry>EDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHK</entry></row><row><entry></entry></row><row><entry>PENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQ</entry></row><row><entry></entry></row><row><entry>LQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK</entry></row><row><entry></entry></row><row><entry>VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER</entry></row><row><entry></entry></row><row><entry>GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKV</entry></row><row><entry></entry></row><row><entry>ITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLE</entry></row><row><entry></entry></row><row><entry>SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANG</entry></row><row><entry></entry></row><row><entry>EIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGF</entry></row><row><entry></entry></row><row><entry>SKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSK</entry></row><row><entry></entry></row><row><entry>KLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE</entry></row><row><entry></entry></row><row><entry>LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQK</entry></row><row><entry></entry></row><row><entry>QLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQ</entry></row><row><entry></entry></row><row><entry>AENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGL</entry></row><row><entry></entry></row><row><entry>YETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIG</entry></row><row><entry></entry></row><row><entry>NKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML</entry></row><row><entry></entry></row><row><entry>SGGSPKKKRKV</entry></row></tbody></tgroup></table></tables><br /> NBE3 (rAPOBEC1-XTEN-Cas9n-UGI-NLS) (SEQ ID NO: 157)

0648<tables id="TABLE-US-00127" num="00127"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYS</entry></row><row><entry></entry></row><row><entry>IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG</entry></row><row><entry></entry></row><row><entry>ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL</entry></row><row><entry></entry></row><row><entry>VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL</entry></row><row><entry></entry></row><row><entry>ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV</entry></row><row><entry></entry></row><row><entry>DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF</entry></row><row><entry></entry></row><row><entry>DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL</entry></row><row><entry></entry></row><row><entry>RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK</entry></row><row><entry></entry></row><row><entry>NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD</entry></row><row><entry></entry></row><row><entry>NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA</entry></row><row><entry></entry></row><row><entry>RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK</entry></row><row><entry></entry></row><row><entry>VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN</entry></row><row><entry></entry></row><row><entry>RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF</entry></row><row><entry></entry></row><row><entry>LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY</entry></row><row><entry></entry></row><row><entry>TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE</entry></row><row><entry></entry></row><row><entry>DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP</entry></row><row><entry></entry></row><row><entry>ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL</entry></row><row><entry></entry></row><row><entry>QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV</entry></row><row><entry></entry></row><row><entry>LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG</entry></row><row><entry></entry></row><row><entry>GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI</entry></row><row><entry></entry></row><row><entry>TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES</entry></row><row><entry></entry></row><row><entry>EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE</entry></row><row><entry></entry></row><row><entry>IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS</entry></row><row><entry></entry></row><row><entry>KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK</entry></row><row><entry></entry></row><row><entry>LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL</entry></row><row><entry></entry></row><row><entry>ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ</entry></row><row><entry></entry></row><row><entry>LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA</entry></row><row><entry></entry></row><row><entry>ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY</entry></row><row><entry></entry></row><row><entry>ETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGN</entry></row><row><entry></entry></row><row><entry>KPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLS</entry></row><row><entry></entry></row><row><entry>GGSPKKKRKV</entry></row></tbody></tgroup></table></tables><br /> pmCDA1-XTEN-dCas9-UGI (Bacteria) (SEQ ID NO: 159)

0649<tables id="TABLE-US-00128" num="00128"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFW</entry></row><row><entry></entry></row><row><entry>GYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADC</entry></row><row><entry></entry></row><row><entry>AEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNV</entry></row><row><entry></entry></row><row><entry>MVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKIL</entry></row><row><entry></entry></row><row><entry>HTTKSPAVSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKV</entry></row><row><entry></entry></row><row><entry>PSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKN</entry></row><row><entry></entry></row><row><entry>RICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVA</entry></row><row><entry></entry></row><row><entry>YHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPD</entry></row><row><entry></entry></row><row><entry>NSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIA</entry></row><row><entry></entry></row><row><entry>QLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN</entry></row><row><entry></entry></row><row><entry>LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE</entry></row><row><entry></entry></row><row><entry>HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIK</entry></row><row><entry></entry></row><row><entry>PILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE</entry></row><row><entry></entry></row><row><entry>DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN</entry></row><row><entry></entry></row><row><entry>FEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKV</entry></row><row><entry></entry></row><row><entry>KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS</entry></row><row><entry></entry></row><row><entry>VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFE</entry></row><row><entry></entry></row><row><entry>DREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK</entry></row><row><entry></entry></row><row><entry>TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL</entry></row><row><entry></entry></row><row><entry>AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNS</entry></row><row><entry></entry></row><row><entry>RERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE</entry></row><row><entry></entry></row><row><entry>LDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVK</entry></row><row><entry></entry></row><row><entry>KMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI</entry></row><row><entry></entry></row><row><entry>TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE</entry></row><row><entry></entry></row><row><entry>INNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ</entry></row><row><entry></entry></row><row><entry>EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG</entry></row><row><entry></entry></row><row><entry>RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD</entry></row><row><entry></entry></row><row><entry>PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN</entry></row><row><entry></entry></row><row><entry>PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA</entry></row><row><entry></entry></row><row><entry>LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS</entry></row><row><entry></entry></row><row><entry>KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF</entry></row><row><entry></entry></row><row><entry>DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSMTNLS</entry></row><row><entry></entry></row><row><entry>DIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENV</entry></row><row><entry></entry></row><row><entry>MLLTSDAPEYKPWALVIQDSNGENKIKML</entry></row></tbody></tgroup></table></tables><br /> pmCDA1-XTEN-nCas9-UGI-NLS (Mammalian Construct) (SEQ ID NO: 160)

0650<tables id="TABLE-US-00129" num="00129"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFW</entry></row><row><entry></entry></row><row><entry>GYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADC</entry></row><row><entry></entry></row><row><entry>AEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNV</entry></row><row><entry></entry></row><row><entry>MVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKIL</entry></row><row><entry></entry></row><row><entry>HTTKSPAVSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKV</entry></row><row><entry></entry></row><row><entry>PSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKN</entry></row><row><entry></entry></row><row><entry>RICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVA</entry></row><row><entry></entry></row><row><entry>YHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPD</entry></row><row><entry></entry></row><row><entry>NSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIA</entry></row><row><entry></entry></row><row><entry>QLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN</entry></row><row><entry></entry></row><row><entry>LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE</entry></row><row><entry></entry></row><row><entry>HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIK</entry></row><row><entry></entry></row><row><entry>PILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE</entry></row><row><entry></entry></row><row><entry>DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN</entry></row><row><entry></entry></row><row><entry>FEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKV</entry></row><row><entry></entry></row><row><entry>KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS</entry></row><row><entry></entry></row><row><entry>VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFE</entry></row><row><entry></entry></row><row><entry>DREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK</entry></row><row><entry></entry></row><row><entry>TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL</entry></row><row><entry></entry></row><row><entry>AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNS</entry></row><row><entry></entry></row><row><entry>RERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE</entry></row><row><entry></entry></row><row><entry>LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVK</entry></row><row><entry></entry></row><row><entry>KMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI</entry></row><row><entry></entry></row><row><entry>TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE</entry></row><row><entry></entry></row><row><entry>INNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ</entry></row><row><entry></entry></row><row><entry>EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG</entry></row><row><entry></entry></row><row><entry>RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD</entry></row><row><entry></entry></row><row><entry>PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN</entry></row><row><entry></entry></row><row><entry>PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA</entry></row><row><entry></entry></row><row><entry>LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS</entry></row><row><entry></entry></row><row><entry>KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF</entry></row><row><entry></entry></row><row><entry>DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSTNLSD</entry></row><row><entry></entry></row><row><entry>IIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVM</entry></row><row><entry></entry></row><row><entry>LLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV</entry></row></tbody></tgroup></table></tables><br /> huAPOBEC3G-XTEN-dCas9-UGI (Bacteria) (SEQ ID NO: 161)

0651<tables id="TABLE-US-00130" num="00130"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQA</entry></row><row><entry></entry></row><row><entry>PHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMA</entry></row><row><entry></entry></row><row><entry>KFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHC</entry></row><row><entry></entry></row><row><entry>WDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQSGSETPGTSESATPES</entry></row><row><entry></entry></row><row><entry>DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL</entry></row><row><entry></entry></row><row><entry>LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL</entry></row><row><entry></entry></row><row><entry>EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL</entry></row><row><entry></entry></row><row><entry>RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI</entry></row><row><entry></entry></row><row><entry>NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN</entry></row><row><entry></entry></row><row><entry>FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL</entry></row><row><entry></entry></row><row><entry>LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF</entry></row><row><entry></entry></row><row><entry>FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK</entry></row><row><entry></entry></row><row><entry>QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY</entry></row><row><entry></entry></row><row><entry>VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN</entry></row><row><entry></entry></row><row><entry>LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL</entry></row><row><entry></entry></row><row><entry>LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII</entry></row><row><entry></entry></row><row><entry>KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL</entry></row><row><entry></entry></row><row><entry>KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS</entry></row><row><entry></entry></row><row><entry>LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM</entry></row><row><entry></entry></row><row><entry>GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV</entry></row><row><entry></entry></row><row><entry>ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDS</entry></row><row><entry></entry></row><row><entry>IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT</entry></row><row><entry></entry></row><row><entry>KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR</entry></row><row><entry></entry></row><row><entry>EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY</entry></row><row><entry></entry></row><row><entry>PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT</entry></row><row><entry></entry></row><row><entry>LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ</entry></row><row><entry></entry></row><row><entry>TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK</entry></row><row><entry></entry></row><row><entry>GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY</entry></row><row><entry></entry></row><row><entry>SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED</entry></row><row><entry></entry></row><row><entry>NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP</entry></row><row><entry></entry></row><row><entry>IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS</entry></row><row><entry></entry></row><row><entry>ITGLYETRIDLSQLGGDSGGSMTNLSDIIEKETGKQLVIQESILMLPEEV</entry></row><row><entry></entry></row><row><entry>EEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGEN</entry></row><row><entry></entry></row><row><entry>KIKML</entry></row></tbody></tgroup></table></tables><br /> huAPOBEC3G-XTEN-nCas9-UGI-NLS (Mammalian Construct) (SEQ ID NO: 162)

0652<tables id="TABLE-US-00131" num="00131"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQA</entry></row><row><entry></entry></row><row><entry>PHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMA</entry></row><row><entry></entry></row><row><entry>KFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHC</entry></row><row><entry></entry></row><row><entry>WDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQSGSETPGTSESATPES</entry></row><row><entry></entry></row><row><entry>DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL</entry></row><row><entry></entry></row><row><entry>LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL</entry></row><row><entry></entry></row><row><entry>EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL</entry></row><row><entry></entry></row><row><entry>RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI</entry></row><row><entry></entry></row><row><entry>NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN</entry></row><row><entry></entry></row><row><entry>FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL</entry></row><row><entry></entry></row><row><entry>LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF</entry></row><row><entry></entry></row><row><entry>FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK</entry></row><row><entry></entry></row><row><entry>QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY</entry></row><row><entry></entry></row><row><entry>VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN</entry></row><row><entry></entry></row><row><entry>LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL</entry></row><row><entry></entry></row><row><entry>LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII</entry></row><row><entry></entry></row><row><entry>KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL</entry></row><row><entry></entry></row><row><entry>KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS</entry></row><row><entry></entry></row><row><entry>LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM</entry></row><row><entry></entry></row><row><entry>GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV</entry></row><row><entry></entry></row><row><entry>ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS</entry></row><row><entry></entry></row><row><entry>IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT</entry></row><row><entry></entry></row><row><entry>KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR</entry></row><row><entry></entry></row><row><entry>EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY</entry></row><row><entry></entry></row><row><entry>PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT</entry></row><row><entry></entry></row><row><entry>LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ</entry></row><row><entry></entry></row><row><entry>TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK</entry></row><row><entry></entry></row><row><entry>GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY</entry></row><row><entry></entry></row><row><entry>SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED</entry></row><row><entry></entry></row><row><entry>NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP</entry></row><row><entry></entry></row><row><entry>IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS</entry></row><row><entry></entry></row><row><entry>ITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVE</entry></row><row><entry></entry></row><row><entry>EVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENK</entry></row><row><entry></entry></row><row><entry>IKMLSGGSPKKKRKV</entry></row></tbody></tgroup></table></tables><br /> huAPOBEC3G (D316R_D317R)-XTEN-nCas9-UGI-NLS (Mammalian Construct) (SEQ ID NO: 163)

0653<tables id="TABLE-US-00132" num="00132"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQA</entry></row><row><entry></entry></row><row><entry>PHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMA</entry></row><row><entry></entry></row><row><entry>KFISKNKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISIMTYSEFKHC</entry></row><row><entry></entry></row><row><entry>WDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQSGSETPGTSESATPES</entry></row><row><entry></entry></row><row><entry>DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL</entry></row><row><entry></entry></row><row><entry>LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL</entry></row><row><entry></entry></row><row><entry>EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL</entry></row><row><entry></entry></row><row><entry>RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI</entry></row><row><entry></entry></row><row><entry>NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN</entry></row><row><entry></entry></row><row><entry>FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL</entry></row><row><entry></entry></row><row><entry>LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF</entry></row><row><entry></entry></row><row><entry>FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK</entry></row><row><entry></entry></row><row><entry>QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY</entry></row><row><entry></entry></row><row><entry>VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN</entry></row><row><entry></entry></row><row><entry>LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL</entry></row><row><entry></entry></row><row><entry>LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII</entry></row><row><entry></entry></row><row><entry>KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL</entry></row><row><entry></entry></row><row><entry>KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS</entry></row><row><entry></entry></row><row><entry>LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM</entry></row><row><entry></entry></row><row><entry>GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV</entry></row><row><entry></entry></row><row><entry>ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS</entry></row><row><entry></entry></row><row><entry>IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT</entry></row><row><entry></entry></row><row><entry>KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR</entry></row><row><entry></entry></row><row><entry>EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY</entry></row><row><entry></entry></row><row><entry>PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT</entry></row><row><entry></entry></row><row><entry>LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ</entry></row><row><entry></entry></row><row><entry>TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK</entry></row><row><entry></entry></row><row><entry>GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY</entry></row><row><entry></entry></row><row><entry>SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED</entry></row><row><entry></entry></row><row><entry>NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP</entry></row><row><entry></entry></row><row><entry>IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS</entry></row><row><entry></entry></row><row><entry>ITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVE</entry></row><row><entry></entry></row><row><entry>EVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENK</entry></row><row><entry></entry></row><row><entry>IKMLSGGSPKKKRKV</entry></row></tbody></tgroup></table></tables>

0654Base Calling Matlab Script

0655<tables id="TABLE-US-00133" num="00133"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 164)</entry></row><row><entry>WTnuc = ‘GCGGACATGGAGGACGTGCGCGGCCGCCTGGTGCAGTACCG</entry></row><row><entry></entry></row><row><entry>CGGCGAGGTGCAGGCCATGCTCGGCCAGA</entry></row><row><entry></entry></row><row><entry>GCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGT</entry></row><row><entry></entry></row><row><entry>AAGCGGCTCCTCCGCGATGCCGATGAC</entry></row><row><entry></entry></row><row><entry>CTGCAGAAGCGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGA</entry></row><row><entry></entry></row><row><entry>GCGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAG’;</entry></row></tbody></tgroup></table></tables>

0656<tables id="TABLE-US-00134" num="00134"><table frame="none" colsep="0" rowsep="0" pgwide="1"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="329pt" align="left" /><thead><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry>%cycle through fastq files for different samples files=dir(‘*.fastq’);</entry></row><row><entry>for d=1:20</entry></row><row><entry>filename=files(d).name;</entry></row><row><entry>%read fastq file</entry></row><row><entry>[header,seqs,qscore]=fastqread(filename);</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="161pt" align="left" /><colspec colname="2" colwidth="168pt" align="left" /><tbody valign="top"><row><entry>seqsLength = length(seqs);</entry><entry>% number of sequences seqsFile=</entry></row><row><entry>strrep(filename,‘.fastq’,‘’);</entry><entry>% trims off .fastq</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="329pt" align="left" /><tbody valign="top"><row><entry>%create a directory with the same name as fastq file ifexist(seqsFile,‘dir’);</entry></row><row><entry>error(‘Directory already exists. Please rename or move it before moving on.’);</entry></row><row><entry>end</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="161pt" align="left" /><colspec colname="2" colwidth="168pt" align="left" /><tbody valign="top"><row><entry>mkdir(seqsFile);</entry><entry>% make directory</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="168pt" align="left" /><colspec colname="2" colwidth="161pt" align="left" /><tbody valign="top"><row><entry>wtLength = length(WTnuc);</entry><entry>% length of wildtype sequence</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="329pt" align="left" /><tbody valign="top"><row><entry>%% aligning back to the wildtype nucleotide sequence</entry></row><row><entry>%</entry></row><row><entry>% AlN is a matrix of the nucleotide alignment window=1:wtLength;</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="154pt" align="left" /><colspec colname="2" colwidth="175pt" align="left" /><tbody valign="top"><row><entry>sBLength = length(seqs);</entry><entry>% number of sequences</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="329pt" align="left" /><tbody valign="top"><row><entry>% counts number of skips nSkips = 0;</entry></row><row><entry>ALN=repmat(‘’,[sBLengthwtLength]);</entry></row><row><entry>% iterate through each sequencing read for i = 1:sBLength</entry></row><row><entry>%If you only have forward read fastq files leave as is</entry></row><row><entry>%If you have R1 foward and R2 is reverse fastq files uncomment the</entry></row><row><entry>%next four lines of code and the subsequent end statement</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="28pt" align="left" /><colspec colname="2" colwidth="301pt" align="left" /><tbody valign="top"><row><entry>%</entry><entry>ifmod(d,2)==0;</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="42pt" align="left" /><colspec colname="2" colwidth="287pt" align="left" /><tbody valign="top"><row><entry>%</entry><entry>reverse=seqrcomplement(seqs{i});</entry></row><row><entry>%</entry><entry>[score,alignment,start]=</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="329pt" align="left" /><tbody valign="top"><row><entry>swalign(reverse,WTnuc,‘Alphabet’,‘NT’);</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="28pt" align="left" /><colspec colname="2" colwidth="301pt" align="left" /><tbody valign="top"><row><entry>%</entry><entry>else</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="329pt" align="left" /><tbody valign="top"><row><entry>[score,alignment,start]=swalign(seqs{i},WTnuc,‘Alphabet’,‘NT’);</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="28pt" align="left" /><colspec colname="2" colwidth="301pt" align="left" /><tbody valign="top"><row><entry>%</entry><entry>end</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="329pt" align="left" /><tbody valign="top"><row><entry>% length of the sequencing read len=</entry></row><row><entry>length(alignment(3,:));</entry></row><row><entry>% if there is a gap in the alignment, skip = 1 and we will</entry></row><row><entry>% throw away the entire read skip = 0;</entry></row><row><entry>for j = 1:len</entry></row><row><entry>if (alignment(3,j) == ‘-’ || alignment(1,j) == ‘-’) skip = 1;</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="offset" colwidth="49pt" align="left" /><colspec colname="1" colwidth="280pt" align="left" /><tbody valign="top"><row><entry /><entry>break;</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="329pt" align="left" /><tbody valign="top"><row><entry>end</entry></row><row><entry>%in addition if the qscore for any given base in the read is</entry></row><row><entry>%below 31 the nucleotide is turned into an N (fastq qscores that are not letters)</entry></row><row><entry>ifisletter(qscore{i}(start(1)+j−1)) else</entry></row><row><entry>alignment(1,j) = ‘N’;</entry></row><row><entry>end</entry></row><row><entry>end</entry></row><row><entry>if skip == 0 && len>10</entry></row><row><entry>ALN(i, start(2):(start(2)+length(alignment)−1))=alignment(1,:);</entry></row><row><entry>    end</entry></row><row><entry>end</entry></row><row><entry>% with the alignment matrices we can simply tally up the occurrences of</entry></row><row><entry>% each nucleotide at each column in the alignment these</entry></row><row><entry>% tallies ignore bases annotated as N</entry></row><row><entry>% due to low qscores</entry></row><row><entry>TallyNTD=zeros(5,wtLength); fori=1:wtLength</entry></row><row><entry>TallyNTD(:,i)=[sum(ALN(:,i)==‘A’),sum(ALN(:,i)==‘C’),sum(ALN(:,i)==‘G’),sum(ALN</entry></row><row><entry>(:,i)==‘T’),sum(ALN(:,i)==‘N’)];</entry></row><row><entry>end</entry></row><row><entry>% we then save these tally matrices in the respective folder for</entry></row><row><entry>% further processing</entry></row><row><entry>save(strcat(seqsFile,‘/TallyNTD’),‘TallyNTD’); dlmwrite(strcat(seqsFile,‘/TallyNTD.txt’),TallyNTD,‘precision’,</entry></row><row><entry>‘%.3f’, ‘newline’, ‘pc’); end</entry></row><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0657INDEL Detection Matlab Script

0658<tables id="TABLE-US-00135" num="00135"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 164)</entry></row><row><entry>WTnuc = ‘GCGGACATGGAGGACGTGCGCGGCCGCCTGGTGCAGTACCGC</entry></row><row><entry></entry></row><row><entry>GGCGAGGTGCAGGCCATGCTCGGCCAGA</entry></row><row><entry></entry></row><row><entry>GCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGTA</entry></row><row><entry></entry></row><row><entry>AGCGGCTCCTCCGCGATGCCGATGAC</entry></row><row><entry></entry></row><row><entry>CTGCAGAAGCGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGAG</entry></row><row><entry></entry></row><row><entry>CGCGGCCTCAGCGCCATCCGCGAGCGCCTGGGGCCCCTGGTGGAACAG’;</entry></row></tbody></tgroup></table></tables>

0659<tables id="TABLE-US-00136" num="00136"><table frame="none" colsep="0" rowsep="0" pgwide="1"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="259pt" align="left" /><thead><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry>%cycle through fastq files for different samples files=dir(‘*.fastq’);</entry></row><row><entry>%specify start and width of indel window as well as length of each flank indelstart=154;</entry></row><row><entry>width=30; flank=10;</entry></row><row><entry>for d=1:3</entry></row><row><entry>filename=files(d).name;</entry></row><row><entry>%read fastq file</entry></row><row><entry>[header,seqs,qscore]=fastqread(filename);</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="126pt" align="left" /><colspec colname="2" colwidth="133pt" align="left" /><tbody valign="top"><row><entry>seqsLength=length(seqs);</entry><entry>% number of sequences seqsFile</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="259pt" align="left" /><tbody valign="top"><row><entry>=strcat(strrep(filename,‘fastq’,‘’),‘_INDELS’);</entry></row><row><entry>%create a directory with the same name as fastq file+_INDELS ifexist(seqsFile,‘dir’);</entry></row><row><entry>error(‘Directory already exists. Please rename or move it before moving on.’);</entry></row><row><entry>end</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="126pt" align="left" /><colspec colname="2" colwidth="133pt" align="left" /><tbody valign="top"><row><entry>mkdir(seqsFile);</entry><entry>% make directory</entry></row><row><entry>wtLength = length(WTnuc);</entry><entry>% length of wildtype sequence sBLength =</entry></row><row><entry>length(seqs);</entry><entry>% number of sequences</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="259pt" align="left" /><tbody valign="top"><row><entry>% initialize counters and cell arrays</entry></row><row><entry>nSkips = 0; notINDEL=0;</entry></row><row><entry>ins={ };</entry></row><row><entry>dels={ }; NumIns=0;</entry></row><row><entry>NumDels=0;</entry></row><row><entry>% iterate through each sequencing read for i = 1:sBLength</entry></row><row><entry>%search for 10BP sequences that should flank both sides of the “INDEL WINDOW”</entry></row><row><entry>windowstart=strfind(seqs{i},WTnuc(indelstart-flank:indelstart));</entry></row><row><entry>     windowend=strfind(seqs{i},WTnuc(indelstart+width:indelstart+width+flank</entry></row><row><entry>));</entry></row><row><entry>%if the flanks are found proceed</entry></row><row><entry>iflength(windowstart)==1 && length(windowend)==1</entry></row><row><entry>%if the sequence length matches the INDEL window length save as</entry></row><row><entry>%not INDEL</entry></row><row><entry>if windowend-windowstart==width+flank notINDEL=notINDEL+1;</entry></row><row><entry>%if the sequence is two or more bases longer than the INDEL</entry></row><row><entry>%window length save as an Insertion</entry></row><row><entry>elseif windowend-windowstart>=width+flank+2 NumIns=NumIns+1;</entry></row><row><entry>ins{NumIns}=seqs{i};</entry></row><row><entry>%if the sequence is two or more bases shorter than the INDEL</entry></row><row><entry>%window length save as a Deletion</entry></row><row><entry>elseif windowend-windowstart<=width+flank-2 NumDels=NumDels+1;</entry></row><row><entry>dels{NumDels}=seqs{i};</entry></row><row><entry>%keep track of skipped sequences that are either one base</entry></row><row><entry>%shorter or longer than the INDEL window width else</entry></row><row><entry>nSkips=nSkips+1;</entry></row><row><entry>end</entry></row><row><entry>%keep track of skipped sequences that do not possess matching flank</entry></row><row><entry>%sequences else</entry></row><row><entry>nSkips=nSkips+1;</entry></row><row><entry>   end</entry></row><row><entry>end</entry></row><row><entry>fid=fopen(strcat(seqsFile,‘/summary.txt’),‘wt’);</entry></row><row><entry>fprintf(fid, ‘Skipped reads %i\n not INDEL %i\n Insertions %i\n Deletions</entry></row><row><entry>%i\n’, [nSkips, notINDEL, NumIns, NumDels]); fclose(fid);</entry></row><row><entry>save(strcat(seqsFile,‘/nSkips’),‘nSkips’); save(strcat(seqsFile,‘/notINDEL’),‘notINDEL’);</entry></row><row><entry>save(strcat(seqsFile,‘/NumIns’),‘NumIns’); save(strcat(seqsFile,‘/NumDels’),‘NumDels’);</entry></row><row><entry>save(strcat(seqsFile,‘/dels’),‘dels’);</entry></row><row><entry>C = dels;</entry></row><row><entry>fid = fopen(strcat(seqsFile, ‘/dels.txt’), ‘wt’); fprintf(fid, ‘“%s”\n’, C{:});</entry></row><row><entry>fclose(fid);</entry></row><row><entry>save(strcat(seqsFile,‘/ins’),‘ins’); C = ins;</entry></row><row><entry>fid = fopen(strcat(seqsFile, ‘/ins.txt’), ‘wt’); fprintf(fid, ‘“%s”\n’, C{:});</entry></row><row><entry>fclose(fid);</entry></row><row><entry>end</entry></row><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

Example 5: Cas9 Variant Sequences

0660The disclosure provides Cas9 variants, for example Cas9 proteins from one or more organisms, which may comprise one or more mutations (e.g., to generate dCas9 or Cas9 nickase). In some embodiments, one or more of the amino acid residues, identified below by an asterisk, of a Cas9 protein may be mutated. In some embodiments, the D10 and/or H840 residues of the amino acid sequence provided in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, are mutated. In some embodiments, the D10 residue of the amino acid sequence provided in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, is mutated to any amino acid residue, except for D. In some embodiments, the D10 residue of the amino acid sequence provided in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, is mutated to an A. In some embodiments, the H840 residue of the amino acid sequence provided in SEQ ID NO: 6, or a corresponding residue in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, is an H. In some embodiments, the H840 residue of the amino acid sequence provided in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, is mutated to any amino acid residue, except for H. In some embodiments, the H840 residue of the amino acid sequence provided in SEQ ID NO: 6, or a corresponding mutation in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, is mutated to an A. In some embodiments, the D10 residue of the amino acid sequence provided in SEQ ID NO: 6, or a corresponding residue in any Cas9 protein, such as any one of the Cas9 amino acid sequences as provided herein, is a D.

0661A number of Cas9 sequences from various species were aligned to determine whether corresponding homologous amino acid residues of D10 and H840 of SEQ ID NO: 6 or SEQ ID NO: 567 can be identified in other Cas9 proteins, allowing the generation of Cas9 variants with corresponding mutations of the homologous amino acid residues. The alignment was carried out using the NCBI Constraint-based Multiple Alignment Tool (COBALT(accessible at st-va.ncbi.nlm.nih.gov/tools/cobalt), with the following parameters. Alignment parameters: Gap penalties −11, −1; End-Gap penalties −5, −1. CDD Parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved columns and Recompute on. Query Clustering Parameters: Use query clusters on; Word Size 4; Max cluster distance 0.8; Alphabet Regular.

0662An exemplary alignment of four Cas9 sequences is provided below. The Cas9 sequences in the alignment are: Sequence 1 (S1): SEQ ID NO: 567|WP_010922251|gi 499224711|type II CRISPR RNA-guided endonuclease Cas9 [<i>Streptococcus pyogenes</i>]; Sequence 2 (S2): SEQ ID NO: 568|WP_039695303|gi 746743737|type II CRISPR RNA-guided endonuclease Cas9 [<i>Streptococcus </i>gallolyticus]; Sequence 3 (S3): SEQ ID NO: 569|WP_045635197|gi 782887988|type II CRISPR RNA-guided endonuclease Cas9 [<i>Streptococcus mitis</i>]; Sequence 4 (S4): SEQ ID NO: 570|5AXW_A|gi 924443546<i>|Staphylococcus aureus </i>Cas9. The HNH domain (bold and underlined) and the RuvC domain (boxed) are identified for each of the four sequences. Amino acid residues 10 and 840 in S1 and the homologous amino acids in the aligned sequences are identified with an asterisk following the respective amino acid residue.

0663<tables id="TABLE-US-00137" num="00137"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="5"><colspec colname="1" colwidth="14pt" align="center" /><colspec colname="2" colwidth="21pt" align="char" /><colspec colname="3" colwidth="343pt" align="left" /><colspec colname="4" colwidth="21pt" align="char" /><colspec colname="5" colwidth="42pt" align="left" /><tbody valign="top"><row><entry>S1</entry><entry>1</entry><entry>--MDKK-<u style="single"><b>YSIGLD*IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI--GALLEDSG--ET</b></u>AEATRLKRTARRRYT</entry><entry>73</entry><entry /></row><row><entry>S2</entry><entry>1</entry><entry>--MTKKN<u style="single"><b>YSIGLD*IGTNSVGWAVITDDYKVPAKKMKVLGNTDKKYIKKNLL--GALLFDSG--ET</b></u>AEATRLKRTARRRYT</entry><entry>74</entry></row><row><entry>S3</entry><entry>1</entry><entry>--M-KKG<u style="single"><b>YSIGLD*IGTNSVGFAVITDDYKVPSKKMKVLGNTDKRFIKKNLI--GALLFDEG--TT</b></u>AEARRLKRTARRRYT</entry><entry>73</entry></row><row><entry>S4</entry><entry>1</entry><entry>GSHMKRN<u style="single"><b>YILGLD*IGITSVGYGII--DYET-----------------RDVIDAGVRLFKEANVEN</b></u>NEGRRSKRGARRLKR</entry><entry>61</entry></row><row><entry></entry></row><row><entry>S1</entry><entry>74</entry><entry>RRKNRICYLQEIFSNEMAKVDDSFEHRLEESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL</entry><entry>153</entry></row><row><entry>S2</entry><entry>75</entry><entry>RRKNRLRYLQEIFANETAKVDESFFQRLDESFLTDDDKTEDSHPIEGNKAEEDAYHQKFPTIYHLRKHLADSSEKADLRL</entry><entry>154</entry></row><row><entry>S3</entry><entry>74</entry><entry>RRKNRLRYLQEIFSEEMSKVDSSFEHRLDDSFLIPEDKRESKYPIFATLTEEKEYHKQFPTIYHLRKQLADSKEKTDLRL</entry><entry>153</entry></row><row><entry>S4</entry><entry>62</entry><entry>RRRHRIQRVKKLL--------------FDYNLLTD--------------------HSELSGINPYEARVKGLSQKLSEEE</entry><entry>107</entry></row><row><entry></entry></row><row><entry>S1</entry><entry>154</entry><entry>IYLALAHMIKERGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEK</entry><entry>233</entry></row><row><entry>S2</entry><entry>155</entry><entry>VYLALAHMIKFRGHFLIEGELNAENTDVQKIFADFVGVYNRTFDDSHLSEITVDVASILTEKISKSRRLENLIKYYPTEK</entry><entry>234</entry></row><row><entry>S3</entry><entry>154</entry><entry>IYLALAHMIKYRGHFLYEEAFDIKNNDIQKIFNEFISIYDNTFEGSSLSGQNAQVEAIFTDKISKSAKRERVLKLEPDEK</entry><entry>233</entry></row><row><entry>S4</entry><entry>108</entry><entry>FSAALLHLAKRRG----------------------VHNVNEVEEDT----------------------------------</entry><entry>131</entry></row><row><entry></entry></row><row><entry>S1</entry><entry>234</entry><entry>KNGLFGNLIALSLGLTPNEKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEIT</entry><entry>313</entry></row><row><entry>S2</entry><entry>235</entry><entry>KNTLFGNLIALALGLQPNEKTNFKLSEDAKLQFSKDTYEEDLEELLGKIGDDYADLFTSAKNLYDAILLSGILTVDDNST</entry><entry>314</entry></row><row><entry>S3</entry><entry>234</entry><entry>STGLFSEFLKLIVGNQADFKKHFDLEDKAPLQFSKDTYDEDLENLLGQIGDDFTDLFVSAKKLYDAILLSGILTVTDPST</entry><entry>313</entry></row><row><entry>S4</entry><entry>132</entry><entry>-----GNELS------------------TKEQISRN--------------------------------------------</entry><entry>144</entry></row><row><entry></entry></row><row><entry>S1</entry><entry>314</entry><entry>KAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKM--DGTEELLV</entry><entry>391</entry></row><row><entry>S2</entry><entry>315</entry><entry>KAPLSASMIKRYVEHHEDLEKLKEFIKANKSELYHDIFKDKNKNGYAGYIENGVKQDEFYKYLKNILSKIKIDGSDYFLD</entry><entry>394</entry></row><row><entry>S3</entry><entry>314</entry><entry>KAPLSASMIERYENHQNDLAALKQFIKNNLPEKYDEVFSDQSKDGYAGYIDGKTTQETFYKYIKNLLSKF--EGTDYFLD</entry><entry>391</entry></row><row><entry>S4</entry><entry>145</entry><entry>----SKALEEKYVAELQ-------------------------------------------------LERLKKDG------</entry><entry>165</entry></row><row><entry></entry></row><row><entry>S1</entry><entry>392</entry><entry>KLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEE</entry><entry>471</entry></row><row><entry>S2</entry><entry>395</entry><entry>KIEREDFLRKQRTFDNGSIPHQIHLQEMHAILRRQGDYYPFLKEKQDRIEKILTFRIPYYVGPLVRKDSRFAWAEYRSDE</entry><entry>474</entry></row><row><entry>S3</entry><entry>392</entry><entry>KIEREDFLRKQRTFDNGSIPHQIHLQEMNAILRRQGEYYPFLKDNKEKIEKILTFRIPYYVGPLARGNRDFAWLTRNSDE</entry><entry>471</entry></row><row><entry>S4</entry><entry>166</entry><entry>--EVRGSINRFKTSD--------YVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGP--GEGSPFGW------K</entry><entry>227</entry></row><row><entry></entry></row><row><entry>S1</entry><entry>472</entry><entry>TITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL</entry><entry>551</entry></row><row><entry>S2</entry><entry>475</entry><entry>KITPWNFDKVIDKEKSAEKFITRMTLNDLYLPEEKVLPKHSHVYETYAVYNELTKIKYVNEQGKE-SFFDSNMKQEIFDH</entry><entry>553</entry></row><row><entry>S3</entry><entry>472</entry><entry>AIRPWNFEEIVDKASSAEDFINKMTNYDLYLPEEKVLPKHSLLYETFAVYNELTKVKFIAEGLRDYQFLDSGQKKQIVNQ</entry><entry>551</entry></row><row><entry>S4</entry><entry>228</entry><entry>DIKEW---------------YEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEK---LEYYEKFQIIEN</entry><entry>289</entry></row><row><entry></entry></row><row><entry>S1</entry><entry>552</entry><entry>LEKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDR---FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED</entry><entry>628</entry></row><row><entry>S2</entry><entry>554</entry><entry>VFKENRKVTKEKLLNYLNKEFPEYRIKDLIGLDKENKSFNASLGTYHDLKKIL-DKAFLDDKVNEEVIEDIIKTLTLFED</entry><entry>632</entry></row><row><entry>S3</entry><entry>552</entry><entry>LEKENRKVTEKDIIHYLHN-VDGYDGIELKGIEKQ---FNASLSTYHDLLKIIKDKEEMDDAKNEAILENIVHTLTIFED</entry><entry>627</entry></row><row><entry>S4</entry><entry>290</entry><entry>VFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEF---TNLKVYHDIKDITARKEII---ENAELLDQIAKILTIYQS</entry><entry>363</entry></row><row><entry></entry></row><row><entry>S1</entry><entry>629</entry><entry>REMIEERLKTYAHLFDDKVMKQLKR-RRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKED</entry><entry>707</entry></row><row><entry>S2</entry><entry>633</entry><entry>KDMIHERLQKYSDIFTANQLKKLER-RHYTGWGRLSYKLINGIRNKENNKTILDYLIDDGSANRNFMQLINDDTLPFKQI</entry><entry>711</entry></row><row><entry>S3</entry><entry>628</entry><entry>REMIKQRLAQYDSLFDEKVIKALTR-RHYTGWGKLSAKLINGICDKQTGNTILDYLIDDGKINRNFMQLINDDGLSFKEI</entry><entry>706</entry></row><row><entry>S4</entry><entry>364</entry><entry>SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDE------LWHTNDNQIAIFNRLKLVP---------</entry><entry>428</entry></row><row><entry></entry></row><row><entry>S1</entry><entry>708</entry><entry><chemistry id="CHEM-US-00242" num="00242"><img file="US11268082B2_D0242.tif" /></chemistry></entry><entry>781</entry></row><row><entry>S2</entry><entry>712</entry><entry><chemistry id="CHEM-US-00243" num="00243"><img file="US11268082B2_D0243.tif" /></chemistry></entry><entry>784</entry></row><row><entry>S3</entry><entry>707</entry><entry><chemistry id="CHEM-US-00244" num="00244"><img file="US11268082B2_D0244.tif" /></chemistry></entry><entry>779</entry></row><row><entry>S4</entry><entry>429</entry><entry><chemistry id="CHEM-US-00245" num="00245"><img file="US11268082B2_D0245.tif" /></chemistry></entry><entry>505</entry></row><row><entry>S1</entry><entry>782</entry><entry><u style="single"><b>KRIEEGIKELGSQIL-------KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD----YDVDH*IVPQSFLKDD</b></u></entry><entry>850</entry></row><row><entry>S2</entry><entry>785</entry><entry><u style="single"><b>KKLQNSLKELGSNILNEEKPSYIEDKVENSHLQNDQLFLYYIQNGKDMYTGDELDIDHLSD----YDIDH*IIPQAFIKDD</b></u></entry><entry>860</entry></row><row><entry>S3</entry><entry>780</entry><entry><u style="single"><b>KRIEDSLKILASGL---DSNILKENPTDNNQLQNDRLFLYYLQNGKDMYTGEALDINQLSS----YDIDH*IIPQAFIKDD</b></u></entry><entry>852</entry></row><row><entry>S4</entry><entry>506</entry><entry><u style="single"><b>ERIEEIIRTTGK---------------ENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDH*IIPRSVSFDN</b></u></entry><entry>570</entry></row><row><entry></entry></row><row><entry>S1</entry><entry>851</entry><entry><chemistry id="CHEM-US-00246" num="00246"><img file="US11268082B2_D0246.tif" /></chemistry></entry><entry>922</entry></row><row><entry>S2</entry><entry>861</entry><entry><chemistry id="CHEM-US-00247" num="00247"><img file="US11268082B2_D0247.tif" /></chemistry></entry><entry>932</entry></row><row><entry>S3</entry><entry>853</entry><entry><chemistry id="CHEM-US-00248" num="00248"><img file="US11268082B2_D0248.tif" /></chemistry></entry><entry>924</entry></row><row><entry>S4</entry><entry>571</entry><entry><chemistry id="CHEM-US-00249" num="00249"><img file="US11268082B2_D0249.tif" /></chemistry></entry><entry>650</entry></row><row><entry>S1</entry><entry>923</entry><entry><chemistry id="CHEM-US-00250" num="00250"><img file="US11268082B2_D0250.tif" /></chemistry></entry><entry>1002</entry></row><row><entry>S2</entry><entry>933</entry><entry><chemistry id="CHEM-US-00251" num="00251"><img file="US11268082B2_D0251.tif" /></chemistry></entry><entry>1012</entry></row><row><entry>S3</entry><entry>925</entry><entry><chemistry id="CHEM-US-00252" num="00252"><img file="US11268082B2_D0252.tif" /></chemistry></entry><entry>1004</entry></row><row><entry>S4</entry><entry>651</entry><entry><chemistry id="CHEM-US-00253" num="00253"><img file="US11268082B2_D0253.tif" /></chemistry></entry><entry>712</entry></row><row><entry>S1</entry><entry>1003</entry><entry><chemistry id="CHEM-US-00254" num="00254"><img file="US11268082B2_D0254.tif" /></chemistry></entry><entry>1077</entry></row><row><entry>S2</entry><entry>1013</entry><entry><chemistry id="CHEM-US-00255" num="00255"><img file="US11268082B2_D0255.tif" /></chemistry></entry><entry>1083</entry></row><row><entry>S3</entry><entry>1005</entry><entry><chemistry id="CHEM-US-00256" num="00256"><img file="US11268082B2_D0256.tif" /></chemistry></entry><entry>1081</entry></row><row><entry>S4</entry><entry>713</entry><entry><chemistry id="CHEM-US-00257" num="00257"><img file="US11268082B2_D0257.tif" /></chemistry></entry><entry>764</entry></row><row><entry>S1</entry><entry>1078</entry><entry><chemistry id="CHEM-US-00258" num="00258"><img file="US11268082B2_D0258.tif" /></chemistry></entry><entry>1149</entry></row><row><entry>S2</entry><entry>1084</entry><entry><chemistry id="CHEM-US-00259" num="00259"><img file="US11268082B2_D0259.tif" /></chemistry></entry><entry>1158</entry></row><row><entry>S3</entry><entry>1082</entry><entry><chemistry id="CHEM-US-00260" num="00260"><img file="US11268082B2_D0260.tif" /></chemistry></entry><entry>1156</entry></row><row><entry>S4</entry><entry>765</entry><entry><chemistry id="CHEM-US-00261" num="00261"><img file="US11268082B2_D0261.tif" /></chemistry></entry><entry>835</entry></row><row><entry>S1</entry><entry>1150</entry><entry>EKGKSKKLKSVKELLGITIMERSSFEKNPI-DFLEAKG-----YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKG</entry><entry>1223</entry></row><row><entry>S2</entry><entry>1159</entry><entry>EKGKAKKLKTVKELVGISIMERSFFEENPV-EFLENKG-----YHNIREDKLIKLPKYSLFEFEGGRRRLLASASELQKG</entry><entry>1232</entry></row><row><entry>S3</entry><entry>1157</entry><entry>EKGKAKKLKTVKTLVGITIMEKAAFEENPI-TFLENKG-----YHNVRKENILCLPKYSLFELENGRRRLLASAKELQKG</entry><entry>1230</entry></row><row><entry>S4</entry><entry>836</entry><entry>DPQTYQKLK--------LIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKV</entry><entry>907</entry></row><row><entry></entry></row><row><entry>S1</entry><entry>1224</entry><entry>NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEITEQISEFSKRVILADANLDKVLSAYNKH------</entry><entry>1297</entry></row><row><entry>S2</entry><entry>1233</entry><entry>NEMVLPGYLVELLYHAHRADNF-----NSTEYLNYVSEHKKEFEKVLSCVEDFANLYVDVEKNLSKIRAVADSM------</entry><entry>1301</entry></row><row><entry>S3</entry><entry>1231</entry><entry>NEIVLPVYLTTLLYHSKNVHKL-----DEPGHLEYIQKHRNEFKDLLNLVSEFSQKYVLADANLEKIKSLYADN------</entry><entry>1299</entry></row><row><entry>S4</entry><entry>908</entry><entry>VKLSLKPYRFD-VYLDNGVYKFV-----TVKNLDVIK--KENYYEVNSKAYEEAKKLKKISNQAEFIASFYNNDLIKING</entry><entry>979</entry></row><row><entry></entry></row><row><entry>S1</entry><entry>1298</entry><entry>RDKPIREQAENITHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSIT--------GLYETRI----DLSQL</entry><entry>1365</entry></row><row><entry>S2</entry><entry>1302</entry><entry>DNFSIEEISNSFINLLTLTALGAPADFNFLGEKIPRKRYTSTKECLNATLIHQSIT--------GLYETRI----DLSKL</entry><entry>1369</entry></row><row><entry>S3</entry><entry>1300</entry><entry>EQADIEILANSFINLLTFTALGAPAAFKFFGKDIDRKRYTTVSEILNATLIHQSIT--------GLYETWI----DLSKL</entry><entry>1367</entry></row><row><entry>S4</entry><entry>980</entry><entry>ELYRVIGVNNDLLNRIEVNMIDITYR-EYLENMNDKRPPRIIKTIASKT---QSIKKYSTDILGNLYEVKSKKHPQIIKK</entry><entry>1055</entry></row><row><entry></entry></row><row><entry>S1</entry><entry>1366</entry><entry>GGD</entry><entry>1368</entry></row><row><entry>S2</entry><entry>1370</entry><entry>GEE</entry><entry>1372</entry></row><row><entry>S3</entry><entry>1368</entry><entry>GED</entry><entry>1370</entry></row><row><entry>S4</entry><entry>1056</entry><entry>G--</entry><entry>1056</entry></row></tbody></tgroup></table></tables>

0664The alignment demonstrates that amino acid sequences and amino acid residues that are homologous to a reference Cas9 amino acid sequence or amino acid residue can be identified across Cas9 sequence variants, including, but not limited to Cas9 sequences from different species, by identifying the amino acid sequence or residue that aligns with the reference sequence or the reference residue using alignment programs and algorithms known in the art. This disclosure provides Cas9 variants in which one or more of the amino acid residues identified by an asterisk in SEQ ID NOs: 567-570 (e.g., S1, S2, S3, and S4, respectively) are mutated as described herein. The residues D10 and H840 in Cas9 of SEQ ID NO: 6 that correspond to the residues identified in SEQ ID NOs: 567-570 by an asterisk are referred to herein as “homologous” or “corresponding” residues. Such homologous residues can be identified by sequence alignment, e.g., as described above, and by identifying the sequence or residue that aligns with the reference sequence or residue. Similarly, mutations in Cas9 sequences that correspond to mutations identified in SEQ ID NO: 6 herein, e.g., mutations of residues 10, and 840 in SEQ ID NO: 6, are referred to herein as “homologous” or “corresponding” mutations. For example, the mutations corresponding to the D10A mutation in SEQ ID NO: 6 or S1 (SEQ ID NO: 567) for the four aligned sequences above are D11A for S2, D10A for S3, and D13A for S4; the corresponding mutations for H840A in SEQ ID NO: 6 or S1 (SEQ ID NO: 567) are H850A for S2, H842A for S3, and H560A for S4.

Example 6: Next Generation C to T Editors

0665Other families of cytidine deaminases as alternatives to base editor 3 (BE3) constructs were examined. The different C to T editors were developed to have a narrow or different editing window, alternate sequence specificity to expand targetable substrates, and to have higher activity.

0666Using the methods described in Example 4, the pmCDA1 (cytidine deaminase 1 from <i>Petromyzon marinus</i>) activity at the HeK-3 site is evaluated (<figref idref="DRAWINGS">FIG. 42</figref>). The pmCDA1-nCas9-UGI-NLS (nCas9 indicates the Cas9 nickase described herein) construct is active on some sites (e.g., the C bases on the complementary strand at position 9, 5, 4, and 3) that are not accessible with rAPOBEC1 (BE3).

0667The pmCDA1 activity at the HeK-2 site is given in <figref idref="DRAWINGS">FIG. 43</figref>. The pmCDA1-XTEN-nCas9-UGI-NLS construct is active on sites adjacent to “G,” while rAPOBEC1 analog (BE3 construct) has low activity on “C”s that are adjacent to “G”s, e.g., the C base at position 11 on the complementary strand.

0668The percent of total sequencing reads with target C converted to T (<figref idref="DRAWINGS">FIG. 44</figref>), C converted to A (<figref idref="DRAWINGS">FIG. 45</figref>), and C converted to G (<figref idref="DRAWINGS">FIG. 46</figref>) are shown for CDA and APOBEC1 (the BE3 construct).

0669The huAPOBEC3G activity at the HeK-2 site is shown in <figref idref="DRAWINGS">FIG. 47</figref>. Two constructs were used: huAPOBEC3G-XTEN-nCas9-UGI-NLS and huAPOBEC3G*(D316R_D317R)-XTEN-nCas9-UGI-NLS. The huAPOBEC3G-XTEN-nCas9-UGI-NLS construct has different sequence specificity than rAPOBEC1 (BE3), as shown in <figref idref="DRAWINGS">FIG. 47</figref>, the editing window appears narrow, as indicated by APOBEC3G's decreased activity at position 4 compared to APOBEC1. Mutations made in huAPOBEC3G (D316R and D317R) increased ssDNA binding and resulted in an observable effect on expanding the sites which were edited (compare APOBEC3G with APOBEC3G_RR in <figref idref="DRAWINGS">FIG. 47</figref>). Mutations were chosen based on APOBEC3G crystal structure, see: Holden et al., Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implication. Nature. (2008); 121-4, the entire contents of which are incorporated herein by reference.

Example 7: pmCDA1/huAPOBEC3G/rAPOBEC1 Work in

E. coli

0670LacZ selection optimization for the A to I conversion was performed using a bacterial strain with lacZ encoded on the F plasmid. A critical glutamic acid residue was mutated (e.g., GAG to GGG, Glu to Gly mutation) so that G to A by a cytidine deaminase would restore lacZ activity (<figref idref="DRAWINGS">FIG. 48</figref>). Strain CC102 was selected for the selection assay. APOBEC1 and CDA constructs were used in a selection assay to optimize G to A conversion.

0671To evaluate the effect of copy number of the plasmids encoding the deaminase constructs on lacZ reversion frequency, the CDA and APOBEC1 deaminases were cloned into 4 plasmids with different replication origins (hence different copy numbers), SC101, CloDF3, RSF1030, and PUC (copy number: PUC>RSF1030>CloDF3>SC101) and placed under an inducible promoter. The plasmids were individually transformed into <i>E. coli </i>cells harboring F plasmid containing the mutated LacZ gene. The expression of the deaminases were induced and LacZ activity was detected for each construct (<figref idref="DRAWINGS">FIG. 49</figref>). As shown in <figref idref="DRAWINGS">FIG. 49</figref>, CDA exhibited significantly higher activity than APOBEC1 in all instances, regardless of the plasmid copy number the deaminases were cloned in. Further, In terms of the copy number, the deaminase activity was positively correlated with the copy number of the plasmid they are cloned in, i.e., PUC>CloDF3>SC101.

0672LacZ reversions were confirmed by sequencing of the genomic DNA at the lacZ locus. To obtain the genomic DNA containing the corrected LacZ gene, cells were grown media containing X-gal, where cells having LacZ activity form blue colonies. Blue colonies were selected and grown in minimal media containing lactose. The cells were spun down, washed, and re-plated on minimal media plates (lactose). The blue colony at the highest dilution was then selected, and its genomic DNA was sequenced at the lacZ locus (<figref idref="DRAWINGS">FIG. 50</figref>).

0673A chloramphenicol reversion assay was designed to test the activity of different cytidine deaminases (e.g., CDA, and APOBEC1). A plasmid harboring a mutant CAT1 gene which confers chloramphenicol resistance to bacteria is constructed with RSF1030 as the replication origin. The mutant CAT1 gene encodings a CAT1 protein that has a H195R (CAC to CGC) mutation, rendering the protein inactive (<figref idref="DRAWINGS">FIG. 51</figref>). Deamination of the C base-paired to the G base in the CGC codon would convert the codon back to a CAC codon, restoring the activity of the protein. As shown in <figref idref="DRAWINGS">FIG. 52</figref>, CDA outperforms rAPOBEC in <i>E. coli </i>in restoring the activity of the chloramphenicol resistance gene. The minimum inhibitory concentration (MIC) of chlor in S1030 with the selection plasmid (pNMG_ch_5) was approximately 1 μg/mL. Both rAPOBEC-XTEN-dCas9-UGI and CDA-XTEN-dCas9-UGI induced DNA correction on the selection plasmid (<figref idref="DRAWINGS">FIG. 53</figref>).

0674Next, the huAPOBEC3G-XTEN-dCas9-UGI protein was tested in the same assay. Interestingly, huAPOBEC3G-XTEN-dCas9-UGI exhibited different sequence specificity than the rAPOBEC1-XTEN-dCas9-UGI fusion protein. Only position 8 was edited with APOBEC3G-XTEN-dCas9-UGI fusion, as compared to the rAPOBEC11-XTEN-dCas9-UGIfusion (in which positions 3, 6, and 8 were edited) (<figref idref="DRAWINGS">FIG. 54</figref>).

Example 8: C to T Base Editors with Less Off Target Editing

0675Current base editing technologies allow for the sequence-specific conversion of a C:G base pair into a T:A base pair in genomic DNA. This is done via the direct catalytic conversion of cytosine to uracil by a cytidine deaminase enzyme and thus, unlike traditional genome editing technologies, does not introduce double-stranded DNA breaks (DSBs) into the DNA as a first step. See, Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., and Liu, D. R. (2016), “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.” Nature 533, 420-424; the entire contents of which are incorporated by reference herein. Instead, catalytically dead SpCas9 (dCas9) or a SpCas9 nickase (dCas9(A840H)) is tethered to a cytidine deaminase enzyme such as rAPOBEC1, pmCDA1, or hAPOBEC3G. The genomic locus of interest is encoded by an sgRNA, and DNA binding and local denaturation is facilitated by the dCas9 portion of the fusion. However, just as wt dCas9 and wt Cas9 exhibit off-target DNA binding and cleavage, current base editors also exhibit C to T editing at Cas9 off-target loci, which limits their therapeutic usefulness.

0676It has been reported that the introduction of just three to four mutations into SpCas9 that neutralize nonspecific electrostatic interactions between the protein and the sugar-phosphate backbone of its target DNA, increases the DNA binding specificity of SpCas9. See, Kleinstiver, B. P., Pattanayak, V., Prew, M. S., Tsai, S. Q., Nguyen, N. T., Zheng, Z., and Joung, J. K. (2016) “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.” Nature 529, 490-495; and Slaymaker, I. M., Gao, L., Zetsche, B., Scott, D. A., Yan, W. X., and Zhang, F. (2015) “Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84-88; the entire contents of each are hereby incorporated by reference herein. Four reported neutralizing mutations were therefore incorporated into the initially reported base editor BE3 (SEQ ID NO: 48), and found that off-target C to T editing of this enzyme is also drastically reduced (<figref idref="DRAWINGS">FIG. 55</figref>), with no decrease in on-target editing (<figref idref="DRAWINGS">FIG. 56</figref>).

0677As shown in <figref idref="DRAWINGS">FIG. 55</figref>, HEK293T cells were transfected with plasmids expressing BE3 or HF-BE3 and a sgRNA matching the EMX1 sequence using LIPOFECTAMINE® 2000 transfection reagent. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target locus, plus the top ten known Cas9 off-target loci for the EMX1 sgRNA, as previously determined by Joung and coworkers using the GUIDE-seq method. See Tsai, S. Q., Zheng, Z., Nguyen, N. T., Liebers, M., Topkar, V. V., Thapar, V., Wyvekens, N., Khayter, C., Iafrate, A. J., Le, L. P., et al. (2015) “GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.” <i>Nat Biotech </i>33, 187-197; the entire contents of which are incorporated by reference herein. EMX1 off-target 5 locus did not amplify and is not shown. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed (<figref idref="DRAWINGS">FIG. 55</figref>). Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for BE3 and HF-BE3.

0678In <figref idref="DRAWINGS">FIG. 56</figref>, HEK293T cells were transfected with plasmids expressing BE3 or HF-BE3 and sgRNAs matching the genomic loci indicated using LIPOFECTAMINE® 2000 transfection reagent. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci. The percentage of total DNA sequencing reads with all four bases at the target Cs within each protospacer are shown for treatment with BE3 or HF-BE3 (<figref idref="DRAWINGS">FIG. 56</figref>). Frequencies of indel formation are shown as well.

0679Primary Protein Sequence of HF-BE3 (SEQ ID NO: 48):

0680<tables id="TABLE-US-00138" num="00138"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>Primary Protein Sequence of HF-BE3:</entry></row><row><entry>(SEQ ID NO: 48)</entry></row><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYS</entry></row><row><entry></entry></row><row><entry>IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG</entry></row><row><entry></entry></row><row><entry>ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL</entry></row><row><entry></entry></row><row><entry>VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL</entry></row><row><entry></entry></row><row><entry>ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV</entry></row><row><entry></entry></row><row><entry>DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF</entry></row><row><entry></entry></row><row><entry>DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL</entry></row><row><entry></entry></row><row><entry>RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK</entry></row><row><entry></entry></row><row><entry>NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD</entry></row><row><entry></entry></row><row><entry>NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA</entry></row><row><entry></entry></row><row><entry>RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEK</entry></row><row><entry></entry></row><row><entry>VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN</entry></row><row><entry></entry></row><row><entry>RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF</entry></row><row><entry></entry></row><row><entry>LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY</entry></row><row><entry></entry></row><row><entry>TGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMALIHDDSLTFKE</entry></row><row><entry></entry></row><row><entry>DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP</entry></row><row><entry></entry></row><row><entry>ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL</entry></row><row><entry></entry></row><row><entry>QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV</entry></row><row><entry></entry></row><row><entry>LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG</entry></row><row><entry></entry></row><row><entry>GLSELDKAGFIKRQLVETRAITKHVAQILDSRMNTKYDENDKLIREVKVI</entry></row><row><entry></entry></row><row><entry>TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES</entry></row><row><entry></entry></row><row><entry>EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE</entry></row><row><entry></entry></row><row><entry>IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS</entry></row><row><entry></entry></row><row><entry>KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK</entry></row><row><entry></entry></row><row><entry>LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL</entry></row><row><entry></entry></row><row><entry>ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ</entry></row><row><entry></entry></row><row><entry>LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA</entry></row><row><entry></entry></row><row><entry>ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY</entry></row><row><entry></entry></row><row><entry>ETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGN</entry></row><row><entry></entry></row><row><entry>KPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLS</entry></row><row><entry></entry></row><row><entry>GGSPKKKRKV</entry></row></tbody></tgroup></table></tables>

Example 9: Development of Base Editors that Use Cas9 Variants and Modulation of the Base Editor Processivity to Increase the Target Range and Precision of the Base Editing Technology

0681Unlike traditional genome editing platforms, base editing technology allows precise single nucleotide changes in the DNA without inducing double-stranded breaks(DSBs). See, Komor, A. C. et al. <i>Nature </i>533, 420-424 (2016). The current generation of base editor uses the NGG PAM exclusively. This limits its ability to edit desired bases within the genome, as the base editor needs to be placed at a precise location where the target base is placed within a 4-base region (the ‘deamination window’), approximately 15 bases upstream of the PAM. See, Komor, A. C. et al. <i>Nature </i>533, 420-424 (2016). Moreover, due to the high processivity of cytidine deaminase, the base editor may convert all cytidines within its deamination window into thymidines, which could induce amino acid changes other than the one desired by the researcher. See, Komor, A. C. et al. <i>Nature </i>533, 420-424 (2016).

0000Expanding the Scope of Base Editing Through the Development of Base Editors with Cas9 Variants

0682Cas9 homologs and other RNA-guided DNA binders that have different PAM specificities were incorporated into the base editor architecture. See, Kleinstiver, B. P. et al. <i>Nature </i>523, 481-485 (2015); Kleinstiver, B. P. et al. <i>Nature Biotechnology </i>33, 1293-1298 (2015); and Zetsche, B. et al. <i>Cell </i>163, 759-771 (2015); the entire contents of each are incorporated by reference herein. Furthermore, innovations that have broadened the PAM specificities of various Cas9 proteins were also incorporated to expand the target reach of the base editor even more. See, Kleinstiver, B. P. et al. <i>Nature </i>523, 481-485 (2015); and Kleinstiver, B. P. et al. <i>Nature Biotechnology </i>33, 1293-1298 (2015). The current palette of base editors is summarized in Table 4.

0683<tables id="TABLE-US-00139" num="00139"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 4</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>New base editors made from Cas9 Variants</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="1" colwidth="42pt" align="left" /><colspec colname="2" colwidth="49pt" align="left" /><colspec colname="3" colwidth="56pt" align="left" /><colspec colname="4" colwidth="70pt" align="left" /><tbody valign="top"><row><entry /><entry /><entry /><entry>Reference for</entry></row><row><entry>Species</entry><entry>PAM</entry><entry>Base Editor Name</entry><entry>Cas9 variant</entry></row><row><entry namest="1" nameend="4" align="center" rowsep="1" /></row><row><entry><i>S. pyogenes</i></entry><entry>. . . NGG</entry><entry>BE3</entry><entry>Wild-type</entry></row><row><entry /><entry>. . . NGA</entry><entry>VQR BE3 or</entry><entry>Kleinstiver, B. P. et al.</entry></row><row><entry /><entry /><entry>EQR BE3</entry></row><row><entry /><entry>. . . NGCG</entry><entry>VRER BE3</entry><entry>Kleinstiver, B. P. et al.</entry></row><row><entry><i>S. aureus</i></entry><entry>. . . NNGRRT</entry><entry>SaBE3</entry><entry>Wild-type</entry></row><row><entry /><entry>. . . NNNRRT</entry><entry>SaKKH BE3</entry><entry>Kleinstiver, B. P. et al.</entry></row><row><entry><i>L. bacterium</i></entry><entry>TTTN . . .</entry><entry>dCpf1 BE2</entry><entry>Zetsche, B. et al.</entry></row><row><entry namest="1" nameend="4" align="center" rowsep="1" /></row></tbody></tgroup></table></tables><br /> Modulating Base Editor's Processivity Through Site-Directed Mutagenesis of rAPOBEC1

0684It was reasoned that the processivity of the base editor could be modulated by making point mutations in the deaminase enzyme. The incorporation of mutations that slightly reduce the catalytic activity of deaminase in which the base editor could still catalyze on average one round of cytidine deamination but was unlikely to access and catalyze another deamination within the relevant timescale were pursued. In effect, the resulting base editor would have a narrower deamination window.

0685rAPOBEC1 mutations probed in this work are listed in Table 5. Some of the mutations resulted in slight apparent impairment of rAPOBEC1 catalysis, which manifested as preferential editing of one cytidine over another when multiple cytidines are found within the deamination window. Combining some of these mutations had an additive effect, allowing the base editor to discriminate substrate cytidines with higher stringency. Some of the double mutants and the triple mutant allowed selective editing of one cytidine among multiple cytidines that are right next to one another (<figref idref="DRAWINGS">FIG. 57</figref>).

0686<tables id="TABLE-US-00140" num="00140"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 5</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>rAPOBEC1 Point Mutations Investigated</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="70pt" align="left" /><colspec colname="2" colwidth="77pt" align="left" /><colspec colname="3" colwidth="70pt" align="left" /><tbody valign="top"><row><entry>rAPOBEC1 mutation</entry><entry>Corresponding mutation</entry><entry /></row><row><entry>studied in this work</entry><entry>in APOBEC3G</entry><entry>Reference</entry></row><row><entry namest="1" nameend="3" align="center" rowsep="1" /></row><row><entry>H121R/H122R</entry><entry>D315R/D316R</entry><entry>Holden, L. G. et al.</entry></row><row><entry>R126A</entry><entry>R320A</entry><entry>Chen, K-M. et al.</entry></row><row><entry>R126E</entry><entry>R320E</entry><entry>Chen, K-M. et al.</entry></row><row><entry>R118A</entry><entry>R313A</entry><entry>Chen, K-M. et al.</entry></row><row><entry>W90A</entry><entry>W285A</entry><entry>Chen, K-M. et al.</entry></row><row><entry>W90Y</entry><entry>W285Y</entry></row><row><entry>R132E</entry><entry>R326E</entry></row><row><entry namest="1" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup></table></tables><br /> Base Editor PAM Expansion and Processivity Modulation

0687The next generation of base editors were designed to expand editable cytidines in the genome by using other RNA-guided DNA binders (<figref idref="DRAWINGS">FIG. 58</figref>). Using a NGG PAM only allows for a single target within the “window” whereas the use of multiple different PAMs allows for Cas9 to be positioned anywhere to effect selective deamination. A variety of new base editors have been created from Cas9 variants (<figref idref="DRAWINGS">FIG. 59</figref> and Table 4). Different PAM sites (NGA, <figref idref="DRAWINGS">FIG. 60</figref>; NGCG, <figref idref="DRAWINGS">FIG. 61</figref>; NNGRRT, <figref idref="DRAWINGS">FIG. 62</figref>; and NNHRRT, <figref idref="DRAWINGS">FIG. 63</figref>) were explored. Selective deamination was successfully achieved through kinetic modulation of cytidine deaminase point mutagenesis (<figref idref="DRAWINGS">FIG. 65</figref> and Table 5).

0688The effect of various mutations on the deamination window was then investigated in cell culture using spacers with multiple cytidines (<figref idref="DRAWINGS">FIGS. 66 and 67</figref>).

0689Further, the effect of various mutations on different genomic sites with limited numbers of cytidines was examined (<figref idref="DRAWINGS">FIGS. 68 to 71</figref>). It was found that approximately one cytidine will be edited within the deamination window in the spacer, while the rest of the cytidines will be left intact. Overall, the preference for editing is as follows: C₆>C₅>>C₇≈C₄.

0000Base Editing Using Cpf1

0690Cpf1, a Cas9 homolog, can be obtained as AsCpf1, LbCpf1, or from any other species. Schematics of fusion constructs, including BE2 and BE3 equivalents, are shown in <figref idref="DRAWINGS">FIG. 73</figref>. The BE2 equivalent uses catalytically inactive Cpf2 enzyme (dCpf1) instead of Cas9, while the BE3 equivalent includes the Cpf1 mutant, which nicks the target strand. The bottom schematic depicts different fusion architectures to combine the two innovations illustrated above it (<figref idref="DRAWINGS">FIG. 73</figref>). The base editing results of HEK293T cell TTTN PAM sites using Cpf1 BE2 were examined with different spacers (<figref idref="DRAWINGS">FIGS. 64A to 64C</figref>). In some embodiments, Cpf1 may be used in place of a Cas9 domain in any of the base editors provided herein. In some embodiments, the Cpf1 is a protein that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% identical to SEQ ID NO 9.

0000Full Protein Sequence of Cpf1 (SEQ ID NO: 9):

0691<tables id="TABLE-US-00141" num="00141"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>Full Protein Sequence of Cpf1:</entry></row><row><entry>(SEQ ID NO: 9)</entry></row><row><entry>MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKA</entry></row><row><entry></entry></row><row><entry>KQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKS</entry></row><row><entry></entry></row><row><entry>AKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGI</entry></row><row><entry></entry></row><row><entry>ELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSII</entry></row><row><entry></entry></row><row><entry>YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKT</entry></row><row><entry></entry></row><row><entry>SEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI</entry></row><row><entry></entry></row><row><entry>NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT</entry></row><row><entry></entry></row><row><entry>TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT</entry></row><row><entry></entry></row><row><entry>DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKY</entry></row><row><entry></entry></row><row><entry>LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLA</entry></row><row><entry></entry></row><row><entry>QISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSED</entry></row><row><entry></entry></row><row><entry>KANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF</entry></row><row><entry></entry></row><row><entry>ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENK</entry></row><row><entry></entry></row><row><entry>GEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN</entry></row><row><entry></entry></row><row><entry>GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSI</entry></row><row><entry></entry></row><row><entry>DEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR</entry></row><row><entry></entry></row><row><entry>PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIA</entry></row><row><entry></entry></row><row><entry>NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEI</entry></row><row><entry></entry></row><row><entry>NLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMK</entry></row><row><entry></entry></row><row><entry>TNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYN</entry></row><row><entry></entry></row><row><entry>AIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG</entry></row><row><entry></entry></row><row><entry>VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYE</entry></row><row><entry></entry></row><row><entry>SVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR</entry></row><row><entry></entry></row><row><entry>LINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD</entry></row><row><entry></entry></row><row><entry>KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNM</entry></row><row><entry></entry></row><row><entry>PQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN</entry></row></tbody></tgroup></table></tables>

Example 10: Increased Fidelity of Base Editing

0692Examining the difference between plasmid delivery of BE3 and HF-BE3, it was found that the two edit on-target loci with comparable efficiency (<figref idref="DRAWINGS">FIGS. 74 and 75</figref>). However, HF-BE3 edited off-target loci much less than BE3, meaning that HF-BE3 has a much higher DNA specificity than BE3 (<figref idref="DRAWINGS">FIG. 76</figref>). Deaminase protein lipofection to HEK cells demonstrated that protein delivery of BE3 results in comparable on-target activity, but much better specificity, than plasmid DNA delivery of BE3. Using improved transfection procedures and better plasmids (n=2), the experiment used the following conditions: protein delivery was 125 nM Cas9:sgRNA complex, plasmid delivery was 750 ng BE3/HF-BE3 plasmid+250 ng sgRNA plasmid, and lipofection was with 1.5 μL of LIPOFECTAMINE® 2000 transfection reagent per well. EMX-1 off target site 2 and FANCF off-target site 1 showed the most off-target editing with BE3, compared to all of the off-targets assayed (<figref idref="DRAWINGS">FIGS. 77 and 78</figref>), while HEK-3 showed no significant editing at off-targets for any of the delivery methods (<figref idref="DRAWINGS">FIG. 79</figref>). HEK-4 shows some C-to-G editing on at the on-target site, while its off-target sites 1, 3, and 4 showed the most off-target editing of all the assayed sites (<figref idref="DRAWINGS">FIG. 80</figref>).

0000Delivery of BE3 Protein Via Micro-Injection to Zebrafish

0693TYR guide RNAs were tested in an in vitro assay for sgRNA activity (<figref idref="DRAWINGS">FIGS. 81 and 82</figref>). The % HTS reads shows how many C residues were converted to T residues during a 2h incubation with purified BE3 protein and PCR of the resulting product. Experiments used an 80-mer synthetic DNA substrate with the target deamination site in 60 bp of its genomic context. This is not the same as % edited DNA strands because only one strand was nicked, so the product is not amplified by PCR. The proportion of HTS reads edited is equal to x/(2-x), where x is the actual proportion of THS reads edited. For 60% editing, the actual proportion of bases edited is 75%. “Off target” is represents BE3 incubated with the same DNA substrate, while bound to an off-target sgRNA. It was found sgRNAs sgRH_13, sgHR_17, and possibly sgHR_16 appeared to be promising targets for in vivo injection experiments.

0694The delivery of BE3 protein in was tested in vivo in zebrafish. Zebrafish embryos (n=16-24) were injected with either scrambled sgRNA, sgHR_13, sgHR_16, or sgHR_17 and purified BE3. Three embryos from each condition were analyzed independently (single embryo) and for each condition, all of the injected embryos were pooled and sequenced as a pool. The results are shown in <figref idref="DRAWINGS">FIGS. 83 to 85</figref>.

Example 11: Uses of Base Editors to Treat Disease

0695Base editors or complexes provided herein (e.g., BE3) may be used to modify nucleic acids. For example, base editors may be used to change a cytosine to a thymine in a nucleic acid (e.g., DNA). Such changes may be made to, inter alia, alter the amino acid sequence of a protein, to destroy or create a start codon, to create a stop codon, to disrupt splicing donors, to disrupt splicing acceptors or edit regulatory sequences. Examples of possible nucleotide changes are shown in <figref idref="DRAWINGS">FIG. 86</figref>.

0696Base editors or complexes provided herein (e.g., BE3) may be used to edit an isoform of Apolipoprotein E in a subject. For example, an Apolipoprotein E isoform may be edited to yield an isoform associated with a lower risk of developing Alzheimer's disease. Apolipoprotein E has four isoforms that differ at amino acids 112 and 158. APOE4 is the largest and most common genetic risk factor for late-onset Alzheimer's disease. Arginine residue 158 of APOE4, encoded by the nucleic acid sequence CGC, may be changed to a cysteine by using a base editor (e.g., BE3) to change the CGC nucleic acid sequence to TGC, which encodes cysteine at residue 158. This change yields an APOE3r isoform, which is associated with lower Alzheimer's disease risk. See <figref idref="DRAWINGS">FIG. 87</figref>.

0697It was tested whether base editor BE3 could be used to edit APOE4 to APOE3r in mouse astrocytes (<figref idref="DRAWINGS">FIG. 88</figref>). APOE 4 mouse astrocytes were nucleofected with Cas9+ template or BE3, targeting the nucleic acid encoding Arginine 158 of APOE4. The Cas9+ template yielded only 0.3% editing with 26% indels, while BE3 yielded 75% editing with 5% indels. Two additional base-edited cytosines are silent and do not yield changes to the amino acid sequence (<figref idref="DRAWINGS">FIG. 88</figref>).

0698Base editors or complexes provided herein may be used to treat prion protein diseases such as Creutzfeldt-Jakob disease and fatal familial insomnia, for example, by introducing mutations into a PRNP gene. Reverting PRNP mutations may not yield therapeutic results, and intels in PRNP may be pathogenic. Accordingly, it was tested whether PRNP could be mutated using base editors (e.g., BE3) to introduce a premature stop codon in the PRNP gene. BE3, associated with its guide RNA, was introduced into HEK cells or glioblastoma cells and was capable of editing the PRNP gene to change the encoded arginine at residue 37 to a stop codon. BE3 yielded 41% editing (<figref idref="DRAWINGS">FIG. 89</figref>).

0699Additional genes that may be edited include the following: APOE editing of Arg 112 and Arg 158 to treat increased Alzheimer's risk; APP editing of Ala 673 to decrease Alzheimer's risk; PRNP editing of Arg 37 to treat fatal familial insomnia and other prion protein diseases; DMD editing of the exons 23 and 51 splice sites to treat Duchenne muscular dystrophy; FTO editing of intron 1 to treat obesity risk; PDS editing of exon 8 to treat Pendred syndrome (genetic deafness); TMC1 editing of exon 8 to treat congenital hearing loss; CYBB editing of various patient-relevant mutations to treat chronic granulomatous disease. Additional diseases that may be treated using the base editors provided herein are shown in Table 6, below.

0700UGI also plays a key role. Knocking out UDG (which UGI inhibits) was shown to dramatically improve the cleanliness and efficiency of C to T base editing (<figref idref="DRAWINGS">FIG. 90</figref>). Furthermore, base editors with nickase and without UGI were shown to produce a mixture of outcomes, with very high indel rates (<figref idref="DRAWINGS">FIG. 91</figref>).

Example 12: Expanding the Targeting Scope of Base Editing

0701Base editing is a new approach to genome editing that uses a fusion protein containing a catalytically defective <i>Streptococcus pyogenes </i>Cas9, a cytidine deaminase, and an inhibitor of base excision repair to induce programmable, single-nucleotide C→T (or G→A) changes in DNA without generating double-strand DNA breaks, without requiring a donor DNA template, and without inducing an excess of stochastic insertions and deletions¹. The development of five new C→T (or G→A) base editors that use natural and engineered Cas9 variants with different protospacer-adjacent motif (PAM) specificities to expand the number of sites that can be targeted by base editing by 2.5-fold are described herein. Additionally, new base editors containing mutated cytidine deaminase domains that narrow the width of the apparent editing window from approximately 5 nucleotides to 1 or 2 nucleotides were engineered, enabling the discrimination of neighboring C nucleotides that would previously be edited with comparable efficiency. Together, these developments substantially increase the targeting scope of base editing.

0702CRISPR-Cas9 nucleases have been widely used to mediate targeted genome editing². In most genome editing applications, Cas9 forms a complex with a single guide RNA (sgRNA) and induces a double-stranded DNA break (DSB) at the target site specified by the sgRNA sequence. Cells primarily respond to this DSB through the non-homolougous end-joining (NHEJ) repair pathway, which results in stochastic insertions or deletions (indels) that can cause frameshift mutations that disrupt the gene. In the presence of a donor DNA template with a high degree of homology to the sequences flanking the DSB, gene correction can be achieved through an alternative pathway known as homology directed repair (HDR).^3,4Unfortunately, under most non-perturbative conditions HDR is inefficient, dependent on cell state and cell type, and dominated by a larger frequency of indels.^3,4As most of the known genetic variations associated with human disease are point mutations⁵, methods that can more efficiently and cleanly make precise point mutations are needed.

0703Base editing, which enables targeted replacement of a C:G base pair with a T:A base pair in a programmable manner without inducing DSBs¹, has been recently described. Base editing uses a fusion protein between a catalytically inactivated (dCas9) or nickase form of <i>Streptococcus pyogenes </i>Cas9 (SpCas9), a cytidine deaminase such as APOBEC1, and an inhibitor of base excision repair such as uracil glycosylase inhibitor (UGI) to convert cytidines into uridines within a five-nucleotide window specified by the sgRNA.¹The third-generation base editor, BE3, converts C:G base pairs to T:A base pairs, including disease-relevant point mutations, in a variety of cell lines with higher efficiency and lower indel frequency than what can be achieved using other genome editing methods¹. Subsequent studies have validated the deaminase-dCas9 fusion approach in a variety of settings^6,7.

0704Efficient editing by BE3 requires the presence of an NGG PAM that places the target C within a five-nucleotide window near the PAM-distal end of the protospacer (positions 4-8, counting the PAM as positions 21-23)¹. This PAM requirement substantially limits the number of sites in the human genome that can be efficiently targeted by BE3, as many sites of interest lack an NGG 13- to 17-nucleotides downstream of the target C. Moreover, the high activity and processivity of BE3 results in conversion of all Cs within the editing window to Ts, which can potentially introduce undesired changes to the target locus. Herein, new C:G to T:A base editors that address both of these limitations are described.

0705It was thought that any Cas9 homolog that binds DNA and forms an “R-loop” complex⁸containing a single-stranded DNA bubble could in principle be converted into a base editor. These new base editors would expand the number of targetable loci by allowing non-NGG PAM sites to be edited. The Cas9 homolog from <i>Staphylococcus aureus </i>(SaCas9) is considerably smaller than SpCas9 (1053 vs. 1368 residues), can mediate efficient genome editing in mammalian cells, and requires an NNGRRT PAM⁹. SpCas9 was replaced with SaCas9 in BE3 to generate SaBE3 and transfected HEK293T cells with plasmids encoding SaBE3 and sgRNAs targeting six human genomic loci (<figref idref="DRAWINGS">FIGS. 92A and 92B</figref>). After 3 d, the genomic loci were subjected to high-throughput DNA sequencing (HTS) to quantify base editing efficiency. SaBE3 enabled C to T base editing of target Cs at a variety of genomic sites in human cells, with very high conversion efficiencies (approximately 50-75% of total DNA sequences converted from C to T, without enrichment for transfected cells) arising from targeting Cs at positions 6-11. The efficiency of SaBE3 on NNGRRT-containing target sites in general exceeded that of BE3 on NGG-containing target sites¹. Perhaps due to its higher average efficiency, SaBE3 can also result in detectable base editing at target Cs at positions outside of the canonical BE3 activity window (<figref idref="DRAWINGS">FIG. 92C</figref>). In comparison, BE3 showed significantly reduced editing under the same conditions (0-11%), in accordance with the known SpCas9 PAM preference (<figref idref="DRAWINGS">FIG. 106A</figref>)¹⁰. These data show that SaBE3 can facilitate very efficient base editing at sites not accessible to BE3.

0706The targeting range of base editors was further expanded by applying recently engineered Cas9 variants that expand or alter PAM specificities. Joung and coworkers recently reported three SpCas9 mutants that accept NGA (VQR-Cas9), NGAG (EQR-Cas9), or NGCG(VRER-Cas9) PAM sequences¹¹. In addition, Joung and coworkers engineered a SaCas9 variant containing three mutations (SaKKH-Cas9) that relax its PAM requirement to NNNRRT¹². The SpCas9 portion of BE3 was replaced with these four Cas9 variants to produce VQR-BE3, EQR-BE3, VRER-BE3, and SaKKH-BE3, which target NNNRRT, NGA, NGAG, and NGCG PAMs respectively. HEK293T cells were transfected with plasmids encoding these constructs and sgRNAs targeting six genomic loci for each new base editor, and measured C to T base conversions using HTS.

0707SaKKH-BE3 edited sites with NNNRRT PAMs with efficiencies up to 62% of treated, non-enriched cells (<figref idref="DRAWINGS">FIG. 92D</figref>). As expected, SaBE3 was unable to efficiently edit targets containing PAMs that were NNNHRRT (where H=A, C, or T) (<figref idref="DRAWINGS">FIG. 92D</figref>). VQR-BE3, EQR-BE3, and VRER-BE3 exhibited more modest, but still substantial base editing efficiencies of up to 50% of treated, non-enriched cells at genomic loci with the expected PAM requirements with an editing window similar to that of BE3 (<figref idref="DRAWINGS">FIGS. 92E and 92F</figref>). Base editing efficiencies of VQR-BE3, EQR-BE3, and VRER-BE3 in general closely paralleled the reported PAM requirements of the corresponding Cas9 nucleases; for example, EQR-BE3 was unable to efficiently edit targets containing NGAH PAM sequences (<figref idref="DRAWINGS">FIG. 92F</figref>). In contrast, BE3 was unable to edit sites with NGA or NGCG PAMs efficiently (0-3%), likely due to its PAM restrictions (<figref idref="DRAWINGS">FIG. 106B</figref>).

0708Collectively, the properties of SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, and VRER-BE3 establish that base editors exhibit a modularity that facilitates their ability to exploit Cas9 homologs and engineered variants.

0709Next, base editors with altered activity window widths were developed. All Cs within the activity window of BE3 can be efficiently converted to Ts¹. The ability to modulate the width of this window would be useful in cases in which it is important to edit only a subset of Cs present in the BE3 activity window.

0710The length of the linker between APOBEC1 and dCas9 was previously observed to modulate the number of bases that are accessible by APOBEC1 in vitro¹. In HEK293T cells, however, varying the linker length did not significantly modulate the width of the editing window, suggesting that in the complex cellular milieu, the relative orientation and flexibility of dCas9 and the cytidine deaminase are not strongly determined by linker length (<figref idref="DRAWINGS">FIG. 96</figref>). Next, it was thought that truncating the 5′ end of the sgRNA might narrow the base editing window by reducing the length of single-stranded DNA accessible to the deaminase upon formation of the RNA-DNA heteroduplex. HEK293T cells were co-transfected with plasmids encoding BE3 and sgRNAs of different spacer lengths targeting a locus with multiple Cs in the editing window. No consistent changes in the width of base editing when using truncated sgRNAs with 17- to 19-base spacers were observed (<figref idref="DRAWINGS">FIGS. 95A to 95C</figref>). Truncating the sgRNA spacer to fewer than 17 bases resulted in large losses in activity (<figref idref="DRAWINGS">FIG. 95A</figref>).

0711As an alternative approach, it was thought that mutations to the deaminase domain might narrow the width of the editing window through multiple possible mechanisms. First, some mutations may alter substrate binding, the conformation of bound DNA, or substrate accessibility to the active site in ways that reduce tolerance for non-optimal presentation of a C to the deaminase active site. Second, because the high activity of APOBEC1 likely contributes to the deamination of multiple Cs per DNA binding event,^1,13,14mutations that reduce the catalytic efficiency of the deaminase domain of a base editor might prevent it from catalyzing successive rounds of deamination before dissociating from the DNA. Once any C:G to T:A editing event has taken place, the sgRNA no longer perfectly matches the target DNA sequence and re-binding of the base editor to the target locus should be less favorable. Both strategies were tested in an effort to discover new base editors that distinguish among multiple cytidines within the original editing window.

0712Given the absence of an available APOBEC1 structure, several mutations previously reported to modulate the catalytic activity of APOBEC3G, a cytidine deaminase from the same family that shares 42% sequence similarity of its active site-containing domain to that of APOBEC1, were identified¹⁵. Corresponding APOBEC1 mutations were incorporated into BE3 and evaluated their effect on base editing efficiency and editing window width in HEK293T cells at two C-rich genomic sites containing Cs at positions 3, 4, 5, 6, 8, 9, 10, 12, 13, and 14 (site A); or containing Cs at positions 5,6, 7, 8, 9, 10, 11, and 13 (site B).

0713The APOBEC1 mutations R118A and W90A each led to dramatic loss of base editing efficiency (<figref idref="DRAWINGS">FIG. 97C</figref>). R132E led to a general decrease in editing efficiency but did not change the substantially narrow the shape of the editing window (<figref idref="DRAWINGS">FIG. 97C</figref>). In contrast, several mutations that narrowed the width of the editing window while maintaining substantial editing efficiency were found (<figref idref="DRAWINGS">FIGS. 93A and 97C</figref>). The “editing window width” was defined to represent the artificially calculated window width within which editing efficiency exceeds the half-maximal value for that target. The editing window width of BE3 for the two C-rich genomic sites tested was 5.0 (site A) and 6.1 (site B) nucleotides.

0714R126 in APOBEC1 is predicted to interact with the phosphate backbone of ssDNA¹³. Previous studies have shown that introducing the corresponding mutation into APOBEC3G decreased catalysis by at least 5-fold¹⁴. Interestingly, when introduced into APOBEC1 in BE3, R126A and R126E increased or maintained activity relative to BE3 at the most strongly edited positions (C5, C6, and C7), while decreasing editing activity at other positions (<figref idref="DRAWINGS">FIGS. 93A and 97C</figref>). Each of these two mutations therefore narrowed the width of the editing window at site A and site B to 4.4 and 3.4 nucleotides (R126A), or to 4.2 and 3.1 nucleotides (R126E), respectively (<figref idref="DRAWINGS">FIGS. 93A and 97C</figref>).

0715W90 in APOBEC1 (corresponding to W285 in APOBEC3G) is predicted to form a hydrophobic pocket in the APOBEC3G active site and assist in substrate binding¹³. Mutating this residue to Ala abrogated APOBEC3G's catalytic activity¹³. In BE3, W90A almost completely abrogated base editing efficiency (<figref idref="DRAWINGS">FIG. 97C</figref>). In contrast, it was found that W90Y only modestly decreased base editing activity while narrowing the editing window width at site A and site B to 3.8 and 4.9 nucleotides, respectively (<figref idref="DRAWINGS">FIG. 93A</figref>). These results demonstrate that mutations to the cytidine deaminase domain can narrow the activity window width of the corresponding base editors.

0716W90Y, R126E, and R132E, the three mutations that narrowed the editing window without drastically reducing base editing activity, were combined into doubly and triply mutated base editors. The double mutant W90Y+R126E resulted in a base editor (YE1-BE3) with BE3-like maximal editing efficiencies, but substantially narrowed editing window width (width at site A and site B=2.9 and 3.0 nucleotides, respectively (<figref idref="DRAWINGS">FIG. 93A</figref>). The W90Y+R132E base editor (YE2-BE3) exhibited modestly lower editing efficiencies (averaging 1.4-fold lower maximal editing yields across the five sites tested compared with BE3), and also substantially narrowed editing window width (width at site A and site B=2.7 and 2.8 nucleotides, respectively) (<figref idref="DRAWINGS">FIG. 97C</figref>). The R126E+R132E double mutant (EE-BE3) showed similar maximal editing efficiencies and editing window width as YE2-BE3 (<figref idref="DRAWINGS">FIG. 97C</figref>). The triple mutant W90Y+R126E+R132E (YEE-BE3) exhibited 2.0-fold lower average maximal editing yields but very little editing beyond the C6 position and an editing window width of 2.1 and 1.4 nucleotides for site A and site B, respectively (<figref idref="DRAWINGS">FIG. 97C</figref>). These data taken together indicate that mutations in the cytidine deaminase domain can strongly affect editing window widths, in some cases with minimal or only modest effects on editing efficiency.

0717The base editing outcomes of BE3, YE1-BE3, YE2-BE3, EE-BE3, and YEE-BE3 were further compared in HEK293T cells targeting four well-studied human genomic sites that contain multiple Cs within the BE3 activity window¹. These target loci contained target Cs at positions 4 and 5 (HEK site 3), positions 4 and 6 (HEK site 2), positions 5 and 6 (EMX1), or positions 6, 7, 8, and 11 (FANCF). BE3 exhibited little (&lt;1.2-fold) preference for editing any Cs within the position 4-8 activity window. In contrast, YE1-BE3, exhibited a 1.3-fold preference for editing C5 over C4 (HEK site 3), 2.6-fold preference for C6 over C4 (HEK site 2), 2.0-fold preference for C5 over C6 (EMX1), and 1.5-fold preference for C6 over C7 (FANCF) (<figref idref="DRAWINGS">FIG. 93B</figref>). YE2-BE3 and EE-BE3 exhibited somewhat greater positional specificity (narrower activity window) than YE1-BE3, averaging 2.4-fold preference for editing C5 over C4 (HEK site 3), 9.5-fold preference for C6 over C4 (HEK site 2), 2.9-fold preference for C5 over C6 (EMX1), and 2.6-fold preference for C7 over C6 (FANCF) (<figref idref="DRAWINGS">FIG. 93B</figref>). YEE-BE3 showed the greatest positional selectivity, with a 2.9-fold preference for editing C5 over C4 (HEK site 3), 29.7-fold preference for C6 over C4 (HEK site 2), 7.9-fold preference for C5 over C6 (EMX1), and 7.9-fold preference for C7 over C6 (FANCF) (<figref idref="DRAWINGS">FIG. 93B</figref>). The findings establish that mutant base editors can discriminate between adjacent Cs, even when both nucleotides are within the BE3 editing window.

0718The product distributions of these four mutants and BE3 were further analyzed by HTS to evaluate their apparent processivity. BE3 generated predominantly T4-T5 (HEK site 3), T4-T6 (HEK site 2), and T5-T6 (EMX1) products in treated HEK293T cells, resulting in, on average, 7.4-fold more products containing two Ts, than products containing a single T. In contrast, YE1-BE3, YE2-BE3, EE-BE3, and YEE-BE3 showed substantially higher preferences for singly edited C4-T5, C4-T6, and T5-C6 products (<figref idref="DRAWINGS">FIG. 93C</figref>). YE1-BE3 yielded products with an average single-T to double-T product ratio of 1.4. YE2-BE3 and EE-BE3 yielded products with an average single-T to double-T product ratio of 4.3 and 5.1, respectively (<figref idref="DRAWINGS">FIG. 93C</figref>). Consistent with the above results, the YEE-BE3 triple mutant favored single-T products by an average of 14.3-fold across the three genomic loci. (<figref idref="DRAWINGS">FIG. 93C</figref>). For the target site in which only one C is within the target window (HEK site 4, at position C5), all four mutants exhibited comparable editing efficiencies as BE3 (<figref idref="DRAWINGS">FIG. 98</figref>). These findings indicate that these BE3 mutants have decreased apparent processivity and can favor the conversion of only a single C at target sites containing multiple Cs within the BE3 editing window. These data also suggest a positional preference of C5>C6>C7≈C4 for these mutant base editors, although this preference could differ depending on the target sequence.

0719The window-modulating mutations in APOBEC1 were applied to VQR-BE3, allowing selective base editing of substrates at sites targeted by NGA PAM (<figref idref="DRAWINGS">FIG. 107A</figref>). However, when these mutations were applied to SaKKH-BE3, a linear decrease in base editing efficiency was observed without the improvement in substrate selectivity, suggesting a different kinetic equilibrium and substrate accessibility of this base editor than those of BE3 and its variants (<figref idref="DRAWINGS">FIG. 107B</figref>).

0720The five base editors with altered PAM specificities described in this study together increase the number of disease-associated mutations in the ClinVar database that can in principle be corrected by base editing by 2.5-fold (<figref idref="DRAWINGS">FIGS. 94A and 94B</figref>). Similarly, the development of base editors with narrowed editing windows approximately doubles the fraction of ClinVar entries with a properly positioned NGG PAM that can be corrected by base editing without comparable modification of a non-target C (from 31% for BE3 to 59% for YEE-BE3) (<figref idref="DRAWINGS">FIGS. 94A and 94B</figref>).

0721In summary, the targeting scope of base editing was substantially expanded by developing base editors that use Cas9 variants with different PAM specificities, and by developing a collection of deaminase mutants with varying editing window widths. In theory, base editing should be possible using other programmable DNA-binding proteins (such as Cpf1¹⁶) that create a bubble of single-stranded DNA that can serve as a substrate for a single-strand-specific nucleotide deaminase enzyme.

0000Materials and Methods

0722Cloning. PCR was performed using Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). Plasmids for BE and sgRNA were constructed using USER cloning (New England Biolabs), obtained from previously reported plasmids¹. DNA vector amplification was carried out using NEB 10beta competent cells (New England Biolabs).

0723Cell culture. HEK293T (ATCC CRL-3216) were cultured in Dulbecco's Modified Eagle's Medium plus GlutaMax (ThermoFisher) supplemented with 10% (v/v) fetal bovine serum (FBS), at 37° C. with 5% CO₂. Immortalized rat astrocytes containing the ApoE4 isoform of the APOE gene (Taconic Biosciences) were maintained in Dulbecco's Modified Eagle's Medium plus GlutaMax (ThermoFisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS) and 200 μg/mL Geneticin (ThermoFisher Scientific).

0724Transfections. HEK293T cells were seeded on 48-well collagen-coated BioCoat plates (Corning) and transfected at approximately 85% confluency. 750 ng of BE and 250 ng of sgRNA expression plasmids were transfected using 1.5 μl of LIPOFECTAMINE® 2000 transfection reagent (ThermoFisher Scientific) per well according to the manufacturer's protocol.

0725High-throughput DNA sequencing of genomic DNA samples. Transfected cells were harvested after 3 d and the genomic DNA was isolated using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter) according to the manufacturer's instructions. Genomic regions of interest were amplified by PCR with flanking HTS primer pairs listed in the Supplementary Sequences. PCR amplification was carried out with Phusion hot-start II DNA polymerase (ThermoFisher) according to the manufacturer's instructions. PCR products were purified using RapidTips (Diffinity Genomics). Secondary PCR was performed to attach sequencing adaptors. The products were gel-purified and quantified using the KAPA Library Quantification Kit-Illumina (KAPA Biosystems). Samples were sequenced on an Illumina MiSeq as previously described¹.

0726Data analysis. Nucleotide frequencies were assessed using a previously described MATLAB script¹. Briefly, the reads were aligned to the reference sequence via the Smith-Waterman algorithm. Base calls with Q-scores below 30 were replaced with a placeholder nucleotide (N). This quality threshold results in nucleotide frequencies with an expected theoretical error rate of 1 in 1000.

0727Analyses of base editing processivity were performed using a custom python script. This program trims sequencing reads to the 20 nucleotide protospacer sequence as determined by a perfect match for the 7 nucleotide sequences that should flank the target site. These targets were then consolidated and sorted by abundance to assess the frequency of base editing products.

0728Bioinformatic analysis of the ClinVar database of human disease-associated mutations was performed in a manner similar to that previously described but with small adjustments¹. These adjustments enable the identification of targets with PAMs of customizable length and sequence. In addition, this improved script includes a priority ranking of target C positions (C5>C6>C7>C8≈C4), thus enabling the identification of target sites in which the on-target C is either the only cytosine within the window or is placed at a position with higher predicted editing efficiency than any off-target C within the editing window.

References for Example 12

0000<ul id="ul0006" list-style="none"><li id="ul0006-0001" num="0729">1 Komor, A. C. et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. <i>Nature </i>533, 420-424 (2016).</li><li id="ul0006-0002" num="0730">2 Sander, J. D. &amp; Joung, J. K. CRISPR-Cas systems for editing, regulating and targeting genomes. <i>Nature biotechnology </i>32, 347-355 (2014).</li><li id="ul0006-0003" num="0731">3 Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. <i>Science </i>339, 819-823 (2013).</li><li id="ul0006-0004" num="0732">4 Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. <i>Nat. Protocols </i>8, 2281-2308 (2013).</li><li id="ul0006-0005" num="0733">Landrum, M. J. et al. ClinVar: public archive of interpretations of clinically relevant variants. <i>Nucleic Acids Res. </i>44, D862-D868 (2015).</li><li id="ul0006-0006" num="0734">6 Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. <i>Science </i>353, aaf8729-1-8 (2016).</li><li id="ul0006-0007" num="0735">7 Ma, Y. et al. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. <i>Nat. Methods </i>doi:10.1038/nmeth.4027 (2016).</li><li id="ul0006-0008" num="0736">8 Jiang, F. et al. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. <i>Science </i>351, 867-71 (2016).</li><li id="ul0006-0009" num="0737">9 Ran, F. A. et al. In vivo genome editing using <i>Staphylococcus aureus </i>Cas9<i>. Nature </i>520, 186-191 (2015).</li><li id="ul0006-0010" num="0738">Zhang, Y. et al. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells. <i>Sci. Rep. </i>4, (2014).</li><li id="ul0006-0011" num="0739">11 Kleinstiver, B. P. et. al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. <i>Nature </i>523, 481-485 (2015).</li><li id="ul0006-0012" num="0740">12 Kleinstiver, B. P. et. al. Broadening the targeting range of <i>Staphylococcus aureus </i>CRISPR-Cas9 by modifying PAM recognition. <i>Nat. Biotechnol. </i>33, 1293-1298 (2015).</li><li id="ul0006-0013" num="0741">13 Holden, L. G. et al. Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications. <i>Nature </i>452, 121-124 (2008).</li><li id="ul0006-0014" num="0742">14 Chen, K.-M. et al. Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G. <i>Nature </i>452, 116-119 (2008).</li><li id="ul0006-0015" num="0743">Harris, R. S., Petersen-Mahrt, S. K. &amp; Neuberger, M. S. RNA Editing Enzyme APOBEC1 and Some of Its Homologs Can Act as DNA Mutators. <i>Molecular Cell </i>10, 1247-1253 (2002).</li><li id="ul0006-0016" num="0744">16 Zetsche, B. et al. Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. <i>Cell </i>163, 759-771 (2015).</li></ul>

Example 13

0745Using improved transfection procedures and better plasmids, biological replicates (n=3) were used to install the four HF mutations into the Cas9 portion of BE3. The mutations do not significantly effect on-targeting editing with plasmid delivery (<figref idref="DRAWINGS">FIG. 99</figref>). At the tested concentration, BE3 protein delivery works; however, the on-target editing is lower than for plasmid delivery (<figref idref="DRAWINGS">FIG. 100</figref>). Protein delivery of BE3 with the HF mutations installed reduces on-targeting editing efficiency but still yields some edited cells (<figref idref="DRAWINGS">FIG. 101</figref>).

0746Both lipofection and installing HF mutations were shown to decrease off-target deamination events. For the four sites shown in <figref idref="DRAWINGS">FIG. 102</figref>, the off-target sites (OT) with the highest GUIDE-Seq reads and deamination events were assayed (Komor et al., Nature, 2016). The specificity ratio was calculated by dividing the off-target editing by the on-target editing at the closest corresponding C. In cases where off-target editing was not detectable, the ratio was set to 100. Thus, a higher specificity ratio indicates a more specific construct. BE3 plasmid delivery showed much higher off-target/on-target editing than protein delivery of BE3, plasmid delivery of HF-BE3, or protein delivery of HF-BE3 (<figref idref="DRAWINGS">FIGS. 102 and 105</figref>).

0747Purified proteins HF-BE3 and BE3 were analyzed in vitro for their capabilities to convert C to T residues at different positions in the spacer with the most permissive motif. Both BE3 and HF-BE3 proteins were found to have the same “window” for base editing (<figref idref="DRAWINGS">FIGS. 103 and 104</figref>).

0748A list of the disease targets is given in Table 8. The base to be edited in Table 8 is indicated in bold and underlined.

0749<tables id="TABLE-US-00142" num="00142"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="315pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 8</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Base Editor Disease Targets</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="8"><colspec colname="1" colwidth="28pt" align="left" /><colspec colname="2" colwidth="63pt" align="left" /><colspec colname="3" colwidth="70pt" align="left" /><colspec colname="4" colwidth="28pt" align="center" /><colspec colname="5" colwidth="21pt" align="left" /><colspec colname="6" colwidth="28pt" align="left" /><colspec colname="7" colwidth="49pt" align="left" /><colspec colname="8" colwidth="28pt" align="left" /><tbody valign="top"><row><entry /><entry /><entry /><entry>SEQ</entry><entry /><entry /><entry /><entry /></row><row><entry>GENE</entry><entry>DISEASE</entry><entry>SPACER</entry><entry>ID NO</entry><entry>PAM</entry><entry>EDITOR</entry><entry>DEFECT</entry><entry>CELL</entry></row><row><entry namest="1" nameend="8" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="8"><colspec colname="1" colwidth="28pt" align="left" /><colspec colname="2" colwidth="63pt" align="left" /><colspec colname="3" colwidth="70pt" align="left" /><colspec colname="4" colwidth="28pt" align="char" char="." /><colspec colname="5" colwidth="21pt" align="left" /><colspec colname="6" colwidth="28pt" align="left" /><colspec colname="7" colwidth="49pt" align="left" /><colspec colname="8" colwidth="28pt" align="left" /><tbody valign="top"><row><entry>RB1</entry><entry>RETINOBLASTOMA</entry><entry>AAT<u style="single"><b>C</b></u>TAGTAAATAA</entry><entry>571</entry><entry>AAAA</entry><entry>SAKKH-</entry><entry>SPLICING</entry><entry>J82</entry></row><row><entry /><entry /><entry>ATTGATGT</entry><entry /><entry>GT</entry><entry>BE3</entry><entry>IMPAIRMENT</entry><entry /></row><row><entry></entry></row><row><entry>PTEN</entry><entry>CANCER</entry><entry>GACCAA<u style="single"><b>C</b></u>GGCTAAG</entry><entry>572</entry><entry>TGA</entry><entry>VQR-</entry><entry>W111R</entry><entry>MC116</entry></row><row><entry /><entry /><entry>TGAAGA</entry><entry /><entry /><entry>BE3</entry><entry /><entry /></row><row><entry></entry></row><row><entry>PIK3CA</entry><entry>CANCER</entry><entry>TC<u style="single"><b>C</b></u>TTTCTTCACGGT</entry><entry>573</entry><entry>ACTG</entry><entry>SAKKH-</entry><entry>K111R</entry><entry>CRL-</entry></row><row><entry /><entry /><entry>TGCCT</entry><entry /><entry>GT</entry><entry>BE3</entry><entry /><entry>5853</entry></row><row><entry></entry></row><row><entry>PIK3CA</entry><entry>CANCER</entry><entry>CTC<u style="single"><b>C</b></u>TGCTCAGTGAT</entry><entry>574</entry><entry>AGA</entry><entry>VQR-</entry><entry>Q546R</entry><entry>CRL-</entry></row><row><entry /><entry /><entry>TTCAG</entry><entry /><entry /><entry>BE3</entry><entry /><entry>2505</entry></row><row><entry></entry></row><row><entry>TP53</entry><entry>CANCER</entry><entry>TGT<u style="single"><b>C</b></u>ACACATGTAGT</entry><entry>575</entry><entry>TGG</entry><entry>YEE-</entry><entry>N239D</entry><entry>SNU475</entry></row><row><entry /><entry /><entry>TGTAG</entry><entry /><entry /><entry>BE3</entry><entry /><entry /></row><row><entry></entry></row><row><entry>HRAS</entry><entry>CANCER</entry><entry>CCTCC<u style="single"><b>C</b></u>GGCCGGCGG</entry><entry>576</entry><entry>AGG</entry><entry>YEE-</entry><entry>Q61R</entry><entry>MC/CAR</entry></row><row><entry /><entry /><entry>TATCC</entry><entry /><entry /><entry>BE3</entry></row><row><entry namest="1" nameend="8" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0750<tables id="TABLE-US-00143" num="00143"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="392pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 6</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Exemplary diseases that may be treated using base editors. The protospacer and PAM</entry></row><row><entry>sequences (SEQ ID NOS: 577-589) are shown in the sgRNA (PAM) column. The PAM</entry></row><row><entry>sequence is shown in parentheses and with the base to be edited indicated by underlining.</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="5"><colspec colname="1" colwidth="119pt" align="left" /><colspec colname="2" colwidth="35pt" align="left" /><colspec colname="3" colwidth="56pt" align="left" /><colspec colname="4" colwidth="133pt" align="left" /><colspec colname="5" colwidth="49pt" align="left" /><tbody valign="top"><row><entry /><entry>gene</entry><entry>Base</entry><entry /><entry /></row><row><entry>Disease target</entry><entry>symbol</entry><entry>changed</entry><entry>sgRNA (PAM)</entry><entry>Base editor</entry></row><row><entry namest="1" nameend="5" align="center" rowsep="1" /></row><row><entry>Prion disease</entry><entry>PRNP</entry><entry>R37*</entry><entry>GGCAGC<u style="single">C</u>GATACCCGGGGCA(GGG)</entry><entry>BE3</entry></row><row><entry /><entry /><entry /><entry>GGGCAGC<u style="single">C</u>GATACCCGGGGC(AGG)</entry><entry /></row><row><entry></entry></row><row><entry>Pendred syndrome</entry><entry>Slc26a4</entry><entry>c.919-2A > G</entry><entry>TTATTGTC<u style="single">C</u>GAAATAAAAGA(AGA)</entry><entry>BE3</entry></row><row><entry /><entry /><entry /><entry>ATTGTC<u style="single">C</u>GAAATAAAAGAAG(AGG)</entry><entry>(VQR</entry></row><row><entry /><entry /><entry /><entry>TTGTC<u style="single">C</u>GAAATAAAAGAAGA(GGA)</entry><entry>SaCas9)</entry></row><row><entry /><entry /><entry /><entry>GTC<u style="single">C</u>GAAATAAAAGAAGAGGAAAA(AAT)</entry><entry /></row><row><entry /><entry /><entry /><entry>GTC<u style="single">C</u>GAAATAAAAGAAGAGGAAAAA(ATT)</entry><entry /></row><row><entry></entry></row><row><entry>Congenital deafness</entry><entry>Tmc1</entry><entry>c.545A > G</entry><entry>CAGGAAG<u style="single">C</u>ACGAGGCCACTG(AGG)</entry><entry>BE3</entry></row><row><entry /><entry /><entry /><entry>AACAGGAAG<u style="single">C</u>ACGAGGCCAC(TGA)</entry><entry>YE-BE3</entry></row><row><entry /><entry /><entry /><entry>AGGAAG<u style="single">C</u>ACGAGGCCACTGA(GGA)</entry><entry>YEE-BE3</entry></row><row><entry></entry></row><row><entry>Acquired deafness</entry><entry>SNHL</entry><entry>S33F</entry><entry>TTGGATT<u style="single">C</u>TGGAATCCATTC(TGG)</entry><entry>BE3</entry></row><row><entry></entry></row><row><entry>Alzheimer's Disease</entry><entry>APP</entry><entry>A673T</entry><entry>TCTGCATCCATCTTCACTTC(AGA)</entry><entry>BE3 VQR</entry></row><row><entry></entry></row><row><entry>Niemann-Pick Disease Type C</entry><entry>NPC1</entry><entry>I1061T</entry><entry>CTTACAGCCAGTAATGTCAC(CGA)</entry><entry>BE3 VQR</entry></row><row><entry namest="1" nameend="5" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

Example 14: Testing Base Editing Constructs

0751Several base editing constructs, including BE3, BE4-pmCDA1, BE4-hAID, BE4-3G, BE4-N, BE4-SSB, BE4-(GGS)₃, BE4-XTEN, BE4-32aa, BE4-2×UGI, and BE4 were tested for their ability to edit a cytosine (C) residue within different target sequences (i.e., EMX1, FANCF, HEK2, HEK3, HEK4, and RNF2). For example, it was tested whether these constructs were capable of producing a C to T mutation. Schematic representations of the base editing constructs are shown in <figref idref="DRAWINGS">FIG. 109</figref>. The target sequences tested are also shown in <figref idref="DRAWINGS">FIG. 109</figref> with the targeted cytosine numbered and indicated in red.

0752The following amino acid sequences were used in the base editing constructs of this example:

UGI:

0753<tables id="TABLE-US-00144" num="00144"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 736)</entry></row><row><entry>TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDEST</entry></row><row><entry></entry></row><row><entry>DENVMLLTSDAPEYKPWALVIQDSNGENKIKML</entry></row></tbody></tgroup></table></tables><br /> rAPOBEC1:

0754<tables id="TABLE-US-00145" num="00145"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 737)</entry></row><row><entry>SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIW</entry></row><row><entry></entry></row><row><entry>RHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAIT</entry></row><row><entry></entry></row><row><entry>EFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGY</entry></row><row><entry></entry></row><row><entry>CWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQP</entry></row><row><entry></entry></row><row><entry>QLTFFTIALQSCHYQRLPPHILWATGLK</entry></row></tbody></tgroup></table></tables><br /> pmCDA1:

0755<tables id="TABLE-US-00146" num="00146"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 81)</entry></row><row><entry>MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFW</entry></row><row><entry></entry></row><row><entry>GYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADC</entry></row><row><entry></entry></row><row><entry>AEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNV</entry></row><row><entry></entry></row><row><entry>MVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKIL</entry></row><row><entry></entry></row><row><entry>HTTKSPAV</entry></row></tbody></tgroup></table></tables><br /> hAID:

0756<tables id="TABLE-US-00147" num="00147"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>SEQ ID NO: 49)</entry></row><row><entry>MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLR</entry></row><row><entry></entry></row><row><entry>NKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG</entry></row><row><entry></entry></row><row><entry>NPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNT</entry></row><row><entry></entry></row><row><entry>FVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL</entry></row></tbody></tgroup></table></tables><br /> hAPOBEC3G:

0757<tables id="TABLE-US-00148" num="00148"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 60)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLA</entry></row><row><entry></entry></row><row><entry>EDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQH</entry></row><row><entry></entry></row><row><entry>CWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNE</entry></row><row><entry></entry></row><row><entry>PWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAE</entry></row><row><entry></entry></row><row><entry>LCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCI</entry></row><row><entry></entry></row><row><entry>FTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQ</entry></row><row><entry></entry></row><row><entry>PWDGLDEHSQDLSGRLRAILQNQEN</entry></row></tbody></tgroup></table></tables><br /> SSB (Single-Stranded DNA Binding Protein):

0758<tables id="TABLE-US-00149" num="00149"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 590)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>ASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKE</entry></row><row><entry></entry></row><row><entry>QTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTE</entry></row><row><entry></entry></row><row><entry>VVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGG</entry></row><row><entry></entry></row><row><entry>AQSRPQQSAPAAPSNEPPMDFDDDIPF</entry></row></tbody></tgroup></table></tables><br /> Linker Sequences: <br /> XTEN:

0759<tables id="TABLE-US-00150" num="00150"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 604)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="offset" colwidth="70pt" align="left" /><colspec colname="1" colwidth="147pt" align="left" /><tbody valign="top"><row><entry /><entry>SGSETPGTSESATPES</entry></row></tbody></tgroup></table></tables><br /> 32aa:

0760<tables id="TABLE-US-00151" num="00151"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 605)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="offset" colwidth="35pt" align="left" /><colspec colname="1" colwidth="182pt" align="left" /><tbody valign="top"><row><entry /><entry>SGGSSGGSSGSETPGTSESATPESSGGSSGGS</entry></row></tbody></tgroup></table></tables><br /> SGGS:

0761<tables id="TABLE-US-00152" num="00152"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 606)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="offset" colwidth="98pt" align="left" /><colspec colname="1" colwidth="119pt" align="left" /><tbody valign="top"><row><entry /><entry>SGGS</entry></row></tbody></tgroup></table></tables><br /> (GGS)₃:

0762<tables id="TABLE-US-00153" num="00153"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 610)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="offset" colwidth="84pt" align="left" /><colspec colname="1" colwidth="133pt" align="left" /><tbody valign="top"><row><entry /><entry>GGSGGSGGS</entry></row></tbody></tgroup></table></tables>

0763The amino acid sequences of the constructs shown in <figref idref="DRAWINGS">FIG. 109</figref> are set forth below:

BE3:

0765<tables id="TABLE-US-00154" num="00154"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 174)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYS</entry></row><row><entry></entry></row><row><entry>IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG</entry></row><row><entry></entry></row><row><entry>ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL</entry></row><row><entry></entry></row><row><entry>VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL</entry></row><row><entry></entry></row><row><entry>ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV</entry></row><row><entry></entry></row><row><entry>DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF</entry></row><row><entry></entry></row><row><entry>DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL</entry></row><row><entry></entry></row><row><entry>RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK</entry></row><row><entry></entry></row><row><entry>NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD</entry></row><row><entry></entry></row><row><entry>NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA</entry></row><row><entry></entry></row><row><entry>RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK</entry></row><row><entry></entry></row><row><entry>VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN</entry></row><row><entry></entry></row><row><entry>RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF</entry></row><row><entry></entry></row><row><entry>LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY</entry></row><row><entry></entry></row><row><entry>TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE</entry></row><row><entry></entry></row><row><entry>DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP</entry></row><row><entry></entry></row><row><entry>ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL</entry></row><row><entry></entry></row><row><entry>QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV</entry></row><row><entry></entry></row><row><entry>LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG</entry></row><row><entry></entry></row><row><entry>GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI</entry></row><row><entry></entry></row><row><entry>TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES</entry></row><row><entry></entry></row><row><entry>EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE</entry></row><row><entry></entry></row><row><entry>IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS</entry></row><row><entry></entry></row><row><entry>KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK</entry></row><row><entry></entry></row><row><entry>LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL</entry></row><row><entry></entry></row><row><entry>ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ</entry></row><row><entry></entry></row><row><entry>LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA</entry></row><row><entry></entry></row><row><entry>ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY</entry></row><row><entry></entry></row><row><entry>ETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGN</entry></row><row><entry></entry></row><row><entry>KPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLS</entry></row><row><entry></entry></row><row><entry>GGSPKKKRKV</entry></row></tbody></tgroup></table></tables><br /> BE4-pmCDA1:

0766<tables id="TABLE-US-00155" num="00155"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 175)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFW</entry></row><row><entry></entry></row><row><entry>GYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADC</entry></row><row><entry></entry></row><row><entry>AEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNV</entry></row><row><entry></entry></row><row><entry>MVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKIL</entry></row><row><entry></entry></row><row><entry>HTTKSPAVSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKV</entry></row><row><entry></entry></row><row><entry>PSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKN</entry></row><row><entry></entry></row><row><entry>RICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVA</entry></row><row><entry></entry></row><row><entry>YHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPD</entry></row><row><entry></entry></row><row><entry>NSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIA</entry></row><row><entry></entry></row><row><entry>QLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN</entry></row><row><entry></entry></row><row><entry>LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE</entry></row><row><entry></entry></row><row><entry>HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIK</entry></row><row><entry></entry></row><row><entry>PILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE</entry></row><row><entry></entry></row><row><entry>DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN</entry></row><row><entry></entry></row><row><entry>FEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKV</entry></row><row><entry></entry></row><row><entry>KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS</entry></row><row><entry></entry></row><row><entry>VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFE</entry></row><row><entry></entry></row><row><entry>DREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK</entry></row><row><entry></entry></row><row><entry>TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL</entry></row><row><entry></entry></row><row><entry>AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNS</entry></row><row><entry></entry></row><row><entry>RERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE</entry></row><row><entry></entry></row><row><entry>LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVK</entry></row><row><entry></entry></row><row><entry>KMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI</entry></row><row><entry></entry></row><row><entry>TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE</entry></row><row><entry></entry></row><row><entry>INNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ</entry></row><row><entry></entry></row><row><entry>EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG</entry></row><row><entry></entry></row><row><entry>RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD</entry></row><row><entry></entry></row><row><entry>PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN</entry></row><row><entry></entry></row><row><entry>PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA</entry></row><row><entry></entry></row><row><entry>LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS</entry></row><row><entry></entry></row><row><entry>KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF</entry></row><row><entry></entry></row><row><entry>DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSTNLSD</entry></row><row><entry></entry></row><row><entry>IIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVM</entry></row><row><entry></entry></row><row><entry>LLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV</entry></row></tbody></tgroup></table></tables>

0767BE4-hAID:

0768<tables id="TABLE-US-00156" num="00156"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 176)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLR</entry></row><row><entry></entry></row><row><entry>NKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG</entry></row><row><entry></entry></row><row><entry>NPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNT</entry></row><row><entry></entry></row><row><entry>FVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGLSG</entry></row><row><entry></entry></row><row><entry>SETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGN</entry></row><row><entry></entry></row><row><entry>TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFS</entry></row><row><entry></entry></row><row><entry>NEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYH</entry></row><row><entry></entry></row><row><entry>LRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQ</entry></row><row><entry></entry></row><row><entry>LVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGL</entry></row><row><entry></entry></row><row><entry>FGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA</entry></row><row><entry></entry></row><row><entry>DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKA</entry></row><row><entry></entry></row><row><entry>LVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTE</entry></row><row><entry></entry></row><row><entry>ELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNR</entry></row><row><entry></entry></row><row><entry>EKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGAS</entry></row><row><entry></entry></row><row><entry>AQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKP</entry></row><row><entry></entry></row><row><entry>AFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRF</entry></row><row><entry></entry></row><row><entry>NASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLK</entry></row><row><entry></entry></row><row><entry>TYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDG</entry></row><row><entry></entry></row><row><entry>FANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI</entry></row><row><entry></entry></row><row><entry>LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEG</entry></row><row><entry></entry></row><row><entry>IKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYD</entry></row><row><entry></entry></row><row><entry>VDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLL</entry></row><row><entry></entry></row><row><entry>NAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDS</entry></row><row><entry></entry></row><row><entry>RMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA</entry></row><row><entry></entry></row><row><entry>YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF</entry></row><row><entry></entry></row><row><entry>FYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVL</entry></row><row><entry></entry></row><row><entry>SMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSP</entry></row><row><entry></entry></row><row><entry>TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGY</entry></row><row><entry></entry></row><row><entry>KEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLY</entry></row><row><entry></entry></row><row><entry>LASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANL</entry></row><row><entry></entry></row><row><entry>DKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYT</entry></row><row><entry></entry></row><row><entry>STKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQL</entry></row><row><entry></entry></row><row><entry>VIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYK</entry></row><row><entry></entry></row><row><entry>PWALVIQDSNGENKIKMLSGGSPKKKRKV</entry></row></tbody></tgroup></table></tables>

BE4-3G:

0770<tables id="TABLE-US-00157" num="00157"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 177)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLA</entry></row><row><entry></entry></row><row><entry>EDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQH</entry></row><row><entry></entry></row><row><entry>CWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNE</entry></row><row><entry></entry></row><row><entry>PWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAE</entry></row><row><entry></entry></row><row><entry>LCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCI</entry></row><row><entry></entry></row><row><entry>FTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQ</entry></row><row><entry></entry></row><row><entry>PWDGLDEHSQDLSGRLRAILQNQENSGSETPGTSESATPESDKKYSIGLA</entry></row><row><entry></entry></row><row><entry>IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE</entry></row><row><entry></entry></row><row><entry>ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEED</entry></row><row><entry></entry></row><row><entry>KKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH</entry></row><row><entry></entry></row><row><entry>MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKA</entry></row><row><entry></entry></row><row><entry>ILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAE</entry></row><row><entry></entry></row><row><entry>DAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT</entry></row><row><entry></entry></row><row><entry>EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA</entry></row><row><entry></entry></row><row><entry>GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSI</entry></row><row><entry></entry></row><row><entry>PHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS</entry></row><row><entry></entry></row><row><entry>RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK</entry></row><row><entry></entry></row><row><entry>HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT</entry></row><row><entry></entry></row><row><entry>VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNE</entry></row><row><entry></entry></row><row><entry>ENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG</entry></row><row><entry></entry></row><row><entry>RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK</entry></row><row><entry></entry></row><row><entry>AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV</entry></row><row><entry></entry></row><row><entry>IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEK</entry></row><row><entry></entry></row><row><entry>LYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRS</entry></row><row><entry></entry></row><row><entry>DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSE</entry></row><row><entry></entry></row><row><entry>LDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS</entry></row><row><entry></entry></row><row><entry>KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVY</entry></row><row><entry></entry></row><row><entry>GDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKR</entry></row><row><entry></entry></row><row><entry>PLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESI</entry></row><row><entry></entry></row><row><entry>LPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV</entry></row><row><entry></entry></row><row><entry>KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR</entry></row><row><entry></entry></row><row><entry>KRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE</entry></row><row><entry></entry></row><row><entry>QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII</entry></row><row><entry></entry></row><row><entry>HLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI</entry></row><row><entry></entry></row><row><entry>DLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPES</entry></row><row><entry></entry></row><row><entry>DILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSP</entry></row><row><entry></entry></row><row><entry>KKKRKV</entry></row></tbody></tgroup></table></tables>

BE4-N:

0772<tables id="TABLE-US-00158" num="00158"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 178)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDES</entry></row><row><entry></entry></row><row><entry>TDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLGGSSSETGPVAVDPTL</entry></row><row><entry></entry></row><row><entry>RRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEV</entry></row><row><entry></entry></row><row><entry>NFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFI</entry></row><row><entry></entry></row><row><entry>YIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE</entry></row><row><entry></entry></row><row><entry>AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCH</entry></row><row><entry></entry></row><row><entry>YQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAV</entry></row><row><entry></entry></row><row><entry>ITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARR</entry></row><row><entry></entry></row><row><entry>RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG</entry></row><row><entry></entry></row><row><entry>NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLI</entry></row><row><entry></entry></row><row><entry>EGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR</entry></row><row><entry></entry></row><row><entry>RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT</entry></row><row><entry></entry></row><row><entry>YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS</entry></row><row><entry></entry></row><row><entry>MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQE</entry></row><row><entry></entry></row><row><entry>EFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH</entry></row><row><entry></entry></row><row><entry>AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSE</entry></row><row><entry></entry></row><row><entry>ETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV</entry></row><row><entry></entry></row><row><entry>YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK</entry></row><row><entry></entry></row><row><entry>KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV</entry></row><row><entry></entry></row><row><entry>LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGI</entry></row><row><entry></entry></row><row><entry>RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL</entry></row><row><entry></entry></row><row><entry>HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT</entry></row><row><entry></entry></row><row><entry>QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGR</entry></row><row><entry></entry></row><row><entry>DMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNV</entry></row><row><entry></entry></row><row><entry>PSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ</entry></row><row><entry></entry></row><row><entry>LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF</entry></row><row><entry></entry></row><row><entry>QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK</entry></row><row><entry></entry></row><row><entry>MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETG</entry></row><row><entry></entry></row><row><entry>EIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI</entry></row><row><entry></entry></row><row><entry>ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME</entry></row><row><entry></entry></row><row><entry>RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL</entry></row><row><entry></entry></row><row><entry>QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEII</entry></row><row><entry></entry></row><row><entry>EQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA</entry></row><row><entry></entry></row><row><entry>PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSG</entry></row><row><entry></entry></row><row><entry>GSPKKKRKV</entry></row></tbody></tgroup></table></tables>

BE4-SSB:

0774<tables id="TABLE-US-00159" num="00159"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 179)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYS</entry></row><row><entry></entry></row><row><entry>IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG</entry></row><row><entry></entry></row><row><entry>ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL</entry></row><row><entry></entry></row><row><entry>VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL</entry></row><row><entry></entry></row><row><entry>ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV</entry></row><row><entry></entry></row><row><entry>DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF</entry></row><row><entry></entry></row><row><entry>DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL</entry></row><row><entry></entry></row><row><entry>RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK</entry></row><row><entry></entry></row><row><entry>NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD</entry></row><row><entry></entry></row><row><entry>NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA</entry></row><row><entry></entry></row><row><entry>RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK</entry></row><row><entry></entry></row><row><entry>VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN</entry></row><row><entry></entry></row><row><entry>RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF</entry></row><row><entry></entry></row><row><entry>LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY</entry></row><row><entry></entry></row><row><entry>TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE</entry></row><row><entry></entry></row><row><entry>DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP</entry></row><row><entry></entry></row><row><entry>ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL</entry></row><row><entry></entry></row><row><entry>QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV</entry></row><row><entry></entry></row><row><entry>LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG</entry></row><row><entry></entry></row><row><entry>GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI</entry></row><row><entry></entry></row><row><entry>TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES</entry></row><row><entry></entry></row><row><entry>EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE</entry></row><row><entry></entry></row><row><entry>IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS</entry></row><row><entry></entry></row><row><entry>KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK</entry></row><row><entry></entry></row><row><entry>LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL</entry></row><row><entry></entry></row><row><entry>ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ</entry></row><row><entry></entry></row><row><entry>LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA</entry></row><row><entry></entry></row><row><entry>ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY</entry></row><row><entry></entry></row><row><entry>ETRIDLSQLGGDSGGSGGSGGSASRGVNKVILVGNLGQDPEVRYMPNGGA</entry></row><row><entry></entry></row><row><entry>VANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVY</entry></row><row><entry></entry></row><row><entry>IEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGG</entry></row><row><entry></entry></row><row><entry>GQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPFS</entry></row><row><entry></entry></row><row><entry>GGSPKKKRKV</entry></row></tbody></tgroup></table></tables>

0775BE4-(GGS)₃:

0776<tables id="TABLE-US-00160" num="00160"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 180)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYS</entry></row><row><entry></entry></row><row><entry>IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG</entry></row><row><entry></entry></row><row><entry>ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL</entry></row><row><entry></entry></row><row><entry>VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL</entry></row><row><entry></entry></row><row><entry>ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV</entry></row><row><entry></entry></row><row><entry>DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF</entry></row><row><entry></entry></row><row><entry>DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL</entry></row><row><entry></entry></row><row><entry>RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK</entry></row><row><entry></entry></row><row><entry>NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD</entry></row><row><entry></entry></row><row><entry>NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA</entry></row><row><entry></entry></row><row><entry>RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK</entry></row><row><entry></entry></row><row><entry>VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN</entry></row><row><entry></entry></row><row><entry>RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF</entry></row><row><entry></entry></row><row><entry>LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY</entry></row><row><entry></entry></row><row><entry>TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE</entry></row><row><entry></entry></row><row><entry>DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP</entry></row><row><entry></entry></row><row><entry>ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL</entry></row><row><entry></entry></row><row><entry>QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV</entry></row><row><entry></entry></row><row><entry>LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG</entry></row><row><entry></entry></row><row><entry>GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI</entry></row><row><entry></entry></row><row><entry>TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES</entry></row><row><entry></entry></row><row><entry>EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE</entry></row><row><entry></entry></row><row><entry>IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS</entry></row><row><entry></entry></row><row><entry>KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK</entry></row><row><entry></entry></row><row><entry>LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL</entry></row><row><entry></entry></row><row><entry>ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ</entry></row><row><entry></entry></row><row><entry>LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA</entry></row><row><entry></entry></row><row><entry>ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY</entry></row><row><entry></entry></row><row><entry>ETRIDLSQLGGDSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEV</entry></row><row><entry></entry></row><row><entry>EEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGEN</entry></row><row><entry></entry></row><row><entry>KIKMLSGGSPKKKRKV</entry></row></tbody></tgroup></table></tables>

BE4-XTEN:

0778<tables id="TABLE-US-00161" num="00161"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 181)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYS</entry></row><row><entry></entry></row><row><entry>IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG</entry></row><row><entry></entry></row><row><entry>ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL</entry></row><row><entry></entry></row><row><entry>VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL</entry></row><row><entry></entry></row><row><entry>ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV</entry></row><row><entry></entry></row><row><entry>DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF</entry></row><row><entry></entry></row><row><entry>DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL</entry></row><row><entry></entry></row><row><entry>RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK</entry></row><row><entry></entry></row><row><entry>NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD</entry></row><row><entry></entry></row><row><entry>NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA</entry></row><row><entry></entry></row><row><entry>RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK</entry></row><row><entry></entry></row><row><entry>VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN</entry></row><row><entry></entry></row><row><entry>RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF</entry></row><row><entry></entry></row><row><entry>LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY</entry></row><row><entry></entry></row><row><entry>TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE</entry></row><row><entry></entry></row><row><entry>DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP</entry></row><row><entry></entry></row><row><entry>ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL</entry></row><row><entry></entry></row><row><entry>QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV</entry></row><row><entry></entry></row><row><entry>LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG</entry></row><row><entry></entry></row><row><entry>GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI</entry></row><row><entry></entry></row><row><entry>TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES</entry></row><row><entry></entry></row><row><entry>EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE</entry></row><row><entry></entry></row><row><entry>IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS</entry></row><row><entry></entry></row><row><entry>KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK</entry></row><row><entry></entry></row><row><entry>LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL</entry></row><row><entry></entry></row><row><entry>ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ</entry></row><row><entry></entry></row><row><entry>LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA</entry></row><row><entry></entry></row><row><entry>ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY</entry></row><row><entry></entry></row><row><entry>ETRIDLSQLGGDSGSETPGTSESATPESTNLSDIIEKETGKQLVIQESIL</entry></row><row><entry></entry></row><row><entry>MLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQ</entry></row><row><entry></entry></row><row><entry>DSNGENKIKMLSGGSPKKKRKV</entry></row></tbody></tgroup></table></tables>

0779BE4-32aa:

0780<tables id="TABLE-US-00162" num="00162"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 182)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGGSSGGSSGSETPGTSESAT</entry></row><row><entry></entry></row><row><entry>PESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR</entry></row><row><entry></entry></row><row><entry>HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM</entry></row><row><entry></entry></row><row><entry>AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRK</entry></row><row><entry></entry></row><row><entry>KLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ</entry></row><row><entry></entry></row><row><entry>TYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN</entry></row><row><entry></entry></row><row><entry>LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLF</entry></row><row><entry></entry></row><row><entry>LAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR</entry></row><row><entry></entry></row><row><entry>QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELL</entry></row><row><entry></entry></row><row><entry>VKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI</entry></row><row><entry></entry></row><row><entry>EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS</entry></row><row><entry></entry></row><row><entry>FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL</entry></row><row><entry></entry></row><row><entry>SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNAS</entry></row><row><entry></entry></row><row><entry>LGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA</entry></row><row><entry></entry></row><row><entry>HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN</entry></row><row><entry></entry></row><row><entry>RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQT</entry></row><row><entry></entry></row><row><entry>VKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE</entry></row><row><entry></entry></row><row><entry>LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH</entry></row><row><entry></entry></row><row><entry>IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK</entry></row><row><entry></entry></row><row><entry>LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMN</entry></row><row><entry></entry></row><row><entry>TKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN</entry></row><row><entry></entry></row><row><entry>AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS</entry></row><row><entry></entry></row><row><entry>NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP</entry></row><row><entry></entry></row><row><entry>QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVA</entry></row><row><entry></entry></row><row><entry>YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEV</entry></row><row><entry></entry></row><row><entry>KKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS</entry></row><row><entry></entry></row><row><entry>HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV</entry></row><row><entry></entry></row><row><entry>LSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTK</entry></row><row><entry></entry></row><row><entry>EVLDATLIHQSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQ</entry></row><row><entry></entry></row><row><entry>ESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWA</entry></row><row><entry></entry></row><row><entry>LVIQDSNGENKIKMLSGGSPKKKRKV</entry></row></tbody></tgroup></table></tables>

BE4-2XUGI:

0782<tables id="TABLE-US-00163" num="00163"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 183)</entry></row><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYS</entry></row><row><entry></entry></row><row><entry>IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG</entry></row><row><entry></entry></row><row><entry>ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL</entry></row><row><entry></entry></row><row><entry>VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL</entry></row><row><entry></entry></row><row><entry>ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV</entry></row><row><entry></entry></row><row><entry>DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF</entry></row><row><entry></entry></row><row><entry>DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL</entry></row><row><entry></entry></row><row><entry>RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK</entry></row><row><entry></entry></row><row><entry>NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD</entry></row><row><entry></entry></row><row><entry>NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA</entry></row><row><entry></entry></row><row><entry>RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK</entry></row><row><entry></entry></row><row><entry>VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN</entry></row><row><entry></entry></row><row><entry>RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF</entry></row><row><entry></entry></row><row><entry>LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY</entry></row><row><entry></entry></row><row><entry>TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE</entry></row><row><entry></entry></row><row><entry>DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP</entry></row><row><entry></entry></row><row><entry>ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL</entry></row><row><entry></entry></row><row><entry>QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV</entry></row><row><entry></entry></row><row><entry>LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG</entry></row><row><entry></entry></row><row><entry>GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI</entry></row><row><entry></entry></row><row><entry>TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES</entry></row><row><entry></entry></row><row><entry>EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE</entry></row><row><entry></entry></row><row><entry>IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS</entry></row><row><entry></entry></row><row><entry>KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK</entry></row><row><entry></entry></row><row><entry>LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL</entry></row><row><entry></entry></row><row><entry>ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ</entry></row><row><entry></entry></row><row><entry>LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA</entry></row><row><entry></entry></row><row><entry>ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY</entry></row><row><entry></entry></row><row><entry>ETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGN</entry></row><row><entry></entry></row><row><entry>KPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLS</entry></row><row><entry></entry></row><row><entry>GGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYD</entry></row><row><entry></entry></row><row><entry>ESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV</entry></row></tbody></tgroup></table></tables>

BE4:

0784<tables id="TABLE-US-00164" num="00164"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 184)</entry></row><row><entry>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</entry></row><row><entry></entry></row><row><entry>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</entry></row><row><entry></entry></row><row><entry>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</entry></row><row><entry></entry></row><row><entry>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ</entry></row><row><entry></entry></row><row><entry>PQLTFFTIALQSCHYQRLPPHILWATGLKSGGSSGGSSGSETPGTSESAT</entry></row><row><entry></entry></row><row><entry>PESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR</entry></row><row><entry></entry></row><row><entry>HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM</entry></row><row><entry></entry></row><row><entry>AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRK</entry></row><row><entry></entry></row><row><entry>KLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ</entry></row><row><entry></entry></row><row><entry>TYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN</entry></row><row><entry></entry></row><row><entry>LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLF</entry></row><row><entry></entry></row><row><entry>LAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR</entry></row><row><entry></entry></row><row><entry>QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELL</entry></row><row><entry></entry></row><row><entry>VKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI</entry></row><row><entry></entry></row><row><entry>EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS</entry></row><row><entry></entry></row><row><entry>FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL</entry></row><row><entry></entry></row><row><entry>SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNAS</entry></row><row><entry></entry></row><row><entry>LGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA</entry></row><row><entry></entry></row><row><entry>HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN</entry></row><row><entry></entry></row><row><entry>RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQT</entry></row><row><entry></entry></row><row><entry>VKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE</entry></row><row><entry></entry></row><row><entry>LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH</entry></row><row><entry></entry></row><row><entry>IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK</entry></row><row><entry></entry></row><row><entry>LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMN</entry></row><row><entry></entry></row><row><entry>TKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN</entry></row><row><entry></entry></row><row><entry>AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS</entry></row><row><entry></entry></row><row><entry>NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP</entry></row><row><entry></entry></row><row><entry>QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVA</entry></row><row><entry></entry></row><row><entry>YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEV</entry></row><row><entry></entry></row><row><entry>KKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS</entry></row><row><entry></entry></row><row><entry>HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV</entry></row><row><entry></entry></row><row><entry>LSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTK</entry></row><row><entry></entry></row><row><entry>EVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSGGSTNLSDIIEKETG</entry></row><row><entry></entry></row><row><entry>KQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAP</entry></row><row><entry></entry></row><row><entry>EYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQE</entry></row><row><entry></entry></row><row><entry>SILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWAL</entry></row><row><entry></entry></row><row><entry>VIQDSNGENKIKMLSGGSPKKKRKV</entry></row></tbody></tgroup></table></tables>

0785The ability of the base editing constructs of <figref idref="DRAWINGS">FIG. 109</figref> to mutate target cytosine residues of EMX1, FANCF, HEK2, HEK3, HEK4, and RNF2 are shown in <figref idref="DRAWINGS">FIGS. 110-115</figref>. The percentage of target cytosines edited (including the proportion of C residues that are mutated to a T, A, or G), as well as the % of indels generated, are shown in <figref idref="DRAWINGS">FIGS. 110-115</figref>. The percentage of target cytosines edited were calculated using the following formula: 100−[% of sequencing reads with C]. The pie charts of <figref idref="DRAWINGS">FIGS. 110-115</figref> show the distribution of reads with the various bases indicated, meaning that looking at all of the base edited reads (reads that have a nucleotide other than a C at the base indicated), what percentage of those have A, G, or T. Tables 9-14, below, show the values of the mutation percentages that are indicated by in the pie charts of <figref idref="DRAWINGS">FIGS. 110-115</figref>.

0786The C to non-T editing observed is likely due to UDG (uracil DNA glycosylase). For example, once the C is converted to the uracil intermediate, UDG can convert it to an abasic site. This abasic site is then processed by other endogenous enzymes and ultimately leads to indels or other bases (such as G or A) replacing the C. We have shown that in UDG knock-out cell lines show increased C to T editing with little to no indels at all.

0787<tables id="TABLE-US-00165" num="00165"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 9</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>EMX1</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="28pt" align="left" /><colspec colname="1" colwidth="91pt" align="left" /><colspec colname="2" colwidth="98pt" align="center" /><tbody valign="top"><row><entry /><entry>EMX1</entry><entry>C₅</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="offset" colwidth="28pt" align="left" /><colspec colname="1" colwidth="77pt" align="left" /><colspec colname="2" colwidth="14pt" align="center" /><colspec colname="3" colwidth="98pt" align="char" char="." /><tbody valign="top"><row><entry /><entry>BE3</entry><entry>A</entry><entry>1.9%</entry></row><row><entry /><entry /><entry>C</entry><entry>51.9%</entry></row><row><entry /><entry /><entry>G</entry><entry>6.0%</entry></row><row><entry /><entry /><entry>T</entry><entry>40.3%</entry></row><row><entry /><entry>pmCDA1</entry><entry>A</entry><entry>0.2%</entry></row><row><entry /><entry /><entry>C</entry><entry>88.9%</entry></row><row><entry /><entry /><entry>G</entry><entry>0.8%</entry></row><row><entry /><entry /><entry>T</entry><entry>10.0%</entry></row><row><entry /><entry>hAID</entry><entry>A</entry><entry>0.3%</entry></row><row><entry /><entry /><entry>C</entry><entry>83.6%</entry></row><row><entry /><entry /><entry>G</entry><entry>0.6%</entry></row><row><entry /><entry /><entry>T</entry><entry>15.4%</entry></row><row><entry /><entry>hAPOBEC3G</entry><entry>A</entry><entry>0.0%</entry></row><row><entry /><entry /><entry>C</entry><entry>98.9%</entry></row><row><entry /><entry /><entry>G</entry><entry>0.2%</entry></row><row><entry /><entry /><entry>T</entry><entry>1.0%</entry></row><row><entry /><entry>BE4-N</entry><entry>A</entry><entry>1.4%</entry></row><row><entry /><entry /><entry>C</entry><entry>85.6%</entry></row><row><entry /><entry /><entry>G</entry><entry>3.5%</entry></row><row><entry /><entry /><entry>T</entry><entry>9.5%</entry></row><row><entry /><entry>BE4-SSB</entry><entry>A</entry><entry>0.3%</entry></row><row><entry /><entry /><entry>C</entry><entry>96.7%</entry></row><row><entry /><entry /><entry>G</entry><entry>0.7%</entry></row><row><entry /><entry /><entry>T</entry><entry>2.3%</entry></row><row><entry /><entry>BE4-(GGS)₃</entry><entry>A</entry><entry>1.7%</entry></row><row><entry /><entry /><entry>C</entry><entry>36.8%</entry></row><row><entry /><entry /><entry>G</entry><entry>4.1%</entry></row><row><entry /><entry /><entry>T</entry><entry>57.4%</entry></row><row><entry /><entry>BE4-XTEN</entry><entry>A</entry><entry>2.1%</entry></row><row><entry /><entry /><entry>C</entry><entry>45.7%</entry></row><row><entry /><entry /><entry>G</entry><entry>5.1%</entry></row><row><entry /><entry /><entry>T</entry><entry>47.1%</entry></row><row><entry /><entry>BE4-32aa</entry><entry>A</entry><entry>1.6%</entry></row><row><entry /><entry /><entry>C</entry><entry>46.3%</entry></row><row><entry /><entry /><entry>G</entry><entry>4.6%</entry></row><row><entry /><entry /><entry>T</entry><entry>47.5%</entry></row><row><entry /><entry>BE4-2XUGI</entry><entry>A</entry><entry>0.6%</entry></row><row><entry /><entry /><entry>C</entry><entry>60.2%</entry></row><row><entry /><entry /><entry>G</entry><entry>1.9%</entry></row><row><entry /><entry /><entry>T</entry><entry>37.3%</entry></row><row><entry /><entry namest="offset" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0788<tables id="TABLE-US-00166" num="00166"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 10</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>FANCF</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="28pt" align="left" /><colspec colname="1" colwidth="91pt" align="left" /><colspec colname="2" colwidth="98pt" align="center" /><tbody valign="top"><row><entry /><entry>FANCF</entry><entry>C₈</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="offset" colwidth="28pt" align="left" /><colspec colname="1" colwidth="77pt" align="left" /><colspec colname="2" colwidth="14pt" align="center" /><colspec colname="3" colwidth="98pt" align="char" char="." /><tbody valign="top"><row><entry /><entry>BE3</entry><entry>A</entry><entry>1.6%</entry></row><row><entry /><entry /><entry>C</entry><entry>74.4%</entry></row><row><entry /><entry /><entry>G</entry><entry>0.7%</entry></row><row><entry /><entry /><entry>T</entry><entry>23.3%</entry></row><row><entry /><entry>pmCDA1</entry><entry>A</entry><entry>0.5%</entry></row><row><entry /><entry /><entry>C</entry><entry>88.8%</entry></row><row><entry /><entry /><entry>G</entry><entry>0.3%</entry></row><row><entry /><entry /><entry>T</entry><entry>10.3%</entry></row><row><entry /><entry>hAID</entry><entry>A</entry><entry>0.3%</entry></row><row><entry /><entry /><entry>C</entry><entry>89.6%</entry></row><row><entry /><entry /><entry>G</entry><entry>0.2%</entry></row><row><entry /><entry /><entry>T</entry><entry>9.9%</entry></row><row><entry /><entry>hAPOBEC3G</entry><entry>A</entry><entry>2.1%</entry></row><row><entry /><entry /><entry>C</entry><entry>75.4%</entry></row><row><entry /><entry /><entry>G</entry><entry>10.4%</entry></row><row><entry /><entry /><entry>T</entry><entry>12.1%</entry></row><row><entry /><entry>BE4-N</entry><entry>A</entry><entry>1.8%</entry></row><row><entry /><entry /><entry>C</entry><entry>83.5%</entry></row><row><entry /><entry /><entry>G</entry><entry>0.6%</entry></row><row><entry /><entry /><entry>T</entry><entry>14.2%</entry></row><row><entry /><entry>BE4-SSB</entry><entry>A</entry><entry>0.3%</entry></row><row><entry /><entry /><entry>C</entry><entry>97.1%</entry></row><row><entry /><entry /><entry>G</entry><entry>0.1%</entry></row><row><entry /><entry /><entry>T</entry><entry>2.6%</entry></row><row><entry /><entry>BE4-(GGS)₃</entry><entry>A</entry><entry>2.3%</entry></row><row><entry /><entry /><entry>C</entry><entry>45.7%</entry></row><row><entry /><entry /><entry>G</entry><entry>1.4%</entry></row><row><entry /><entry /><entry>T</entry><entry>50.6%</entry></row><row><entry /><entry>BE4-XTEN</entry><entry>A</entry><entry>1.1%</entry></row><row><entry /><entry /><entry>C</entry><entry>56.3%</entry></row><row><entry /><entry /><entry>G</entry><entry>0.4%</entry></row><row><entry /><entry /><entry>T</entry><entry>42.1%</entry></row><row><entry /><entry>BE4-32aa</entry><entry>A</entry><entry>1.2%</entry></row><row><entry /><entry /><entry>C</entry><entry>70.0%</entry></row><row><entry /><entry /><entry>G</entry><entry>0.6%</entry></row><row><entry /><entry /><entry>T</entry><entry>28.2%</entry></row><row><entry /><entry>BE4-2XUGI</entry><entry>A</entry><entry>0.9%</entry></row><row><entry /><entry /><entry>C</entry><entry>57.8%</entry></row><row><entry /><entry /><entry>G</entry><entry>1.1%</entry></row><row><entry /><entry /><entry>T</entry><entry>40.2%</entry></row><row><entry /><entry namest="offset" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0789<tables id="TABLE-US-00167" num="00167"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 11</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>HEK2</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="28pt" align="left" /><colspec colname="1" colwidth="91pt" align="left" /><colspec colname="2" colwidth="98pt" align="center" /><tbody valign="top"><row><entry /><entry>HEK2</entry><entry>C₆</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="offset" colwidth="28pt" align="left" /><colspec colname="1" colwidth="77pt" align="left" /><colspec colname="2" colwidth="14pt" align="center" /><colspec colname="3" colwidth="98pt" align="char" char="." /><tbody valign="top"><row><entry /><entry>BE3</entry><entry>A</entry><entry>0.9%</entry></row><row><entry /><entry /><entry>C</entry><entry>28.2%</entry></row><row><entry /><entry /><entry>G</entry><entry>52.9%</entry></row><row><entry /><entry /><entry>T</entry><entry>18.1%</entry></row><row><entry /><entry>pmCDA1</entry><entry>A</entry><entry>1.8%</entry></row><row><entry /><entry /><entry>C</entry><entry>73.5%</entry></row><row><entry /><entry /><entry>G</entry><entry>3.5%</entry></row><row><entry /><entry /><entry>T</entry><entry>21.2%</entry></row><row><entry /><entry>hAID</entry><entry>A</entry><entry>2.1%</entry></row><row><entry /><entry /><entry>C</entry><entry>56.9%</entry></row><row><entry /><entry /><entry>G</entry><entry>7.7%</entry></row><row><entry /><entry /><entry>T</entry><entry>33.3%</entry></row><row><entry /><entry>hAPOBEC3G</entry><entry>A</entry><entry>0.1%</entry></row><row><entry /><entry /><entry>C</entry><entry>86.5%</entry></row><row><entry /><entry /><entry>G</entry><entry>9.9%</entry></row><row><entry /><entry /><entry>T</entry><entry>3.5%</entry></row><row><entry /><entry>BE4-N</entry><entry>A</entry><entry>1.0%</entry></row><row><entry /><entry /><entry>C</entry><entry>37.6%</entry></row><row><entry /><entry /><entry>G</entry><entry>57.0%</entry></row><row><entry /><entry /><entry>T</entry><entry>4.4%</entry></row><row><entry /><entry>BE4-SSB</entry><entry>A</entry><entry>0.2%</entry></row><row><entry /><entry /><entry>C</entry><entry>78.4%</entry></row><row><entry /><entry /><entry>G</entry><entry>20.0%</entry></row><row><entry /><entry /><entry>T</entry><entry>1.4%</entry></row><row><entry /><entry>BE4-(GGS)₃</entry><entry>A</entry><entry>0.6%</entry></row><row><entry /><entry /><entry>C</entry><entry>11.1%</entry></row><row><entry /><entry /><entry>G</entry><entry>40.6%</entry></row><row><entry /><entry /><entry>T</entry><entry>47.7%</entry></row><row><entry /><entry>BE4-XTEN</entry><entry>A</entry><entry>1.2%</entry></row><row><entry /><entry /><entry>C</entry><entry>24.8%</entry></row><row><entry /><entry /><entry>G</entry><entry>44.6%</entry></row><row><entry /><entry /><entry>T</entry><entry>29.4%</entry></row><row><entry /><entry>BE4-32aa</entry><entry>A</entry><entry>1.1%</entry></row><row><entry /><entry /><entry>C</entry><entry>26.3%</entry></row><row><entry /><entry /><entry>G</entry><entry>41.8%</entry></row><row><entry /><entry /><entry>T</entry><entry>30.7%</entry></row><row><entry /><entry>BE4-2XUGI</entry><entry>A</entry><entry>0.8%</entry></row><row><entry /><entry /><entry>C</entry><entry>37.0%</entry></row><row><entry /><entry /><entry>G</entry><entry>21.6%</entry></row><row><entry /><entry /><entry>T</entry><entry>40.6%</entry></row><row><entry /><entry namest="offset" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0790<tables id="TABLE-US-00168" num="00168"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 12</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>HEK3</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="35pt" align="left" /><colspec colname="1" colwidth="91pt" align="left" /><colspec colname="2" colwidth="91pt" align="center" /><tbody valign="top"><row><entry /><entry>HEK3</entry><entry>C₅</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="offset" colwidth="35pt" align="left" /><colspec colname="1" colwidth="77pt" align="left" /><colspec colname="2" colwidth="14pt" align="center" /><colspec colname="3" colwidth="91pt" align="center" /><tbody valign="top"><row><entry /><entry>BE3</entry><entry>A</entry><entry>2.23%</entry></row><row><entry /><entry /><entry>C</entry><entry>38.06% </entry></row><row><entry /><entry /><entry>G</entry><entry>12.77% </entry></row><row><entry /><entry /><entry>T</entry><entry>46.95% </entry></row><row><entry /><entry>pmCDA1</entry><entry>A</entry><entry>0.21%</entry></row><row><entry /><entry /><entry>C</entry><entry>76.57% </entry></row><row><entry /><entry /><entry>G</entry><entry>0.12%</entry></row><row><entry /><entry /><entry>T</entry><entry>23.09% </entry></row><row><entry /><entry>hAID</entry><entry>A</entry><entry>0.28%</entry></row><row><entry /><entry /><entry>C</entry><entry>60.23% </entry></row><row><entry /><entry /><entry>G</entry><entry>1.03%</entry></row><row><entry /><entry /><entry>T</entry><entry>38.45% </entry></row><row><entry /><entry>hAPOBEC3G</entry><entry>A</entry><entry>3.11%</entry></row><row><entry /><entry /><entry>C</entry><entry>33.89% </entry></row><row><entry /><entry /><entry>G</entry><entry>28.59% </entry></row><row><entry /><entry /><entry>T</entry><entry>34.41% </entry></row><row><entry /><entry>BE4-N</entry><entry>A</entry><entry> 2.6%</entry></row><row><entry /><entry /><entry>C</entry><entry>64.1%</entry></row><row><entry /><entry /><entry>G</entry><entry>13.5%</entry></row><row><entry /><entry /><entry>T</entry><entry>19.8%</entry></row><row><entry /><entry>BE4-SSB</entry><entry>A</entry><entry> 0.4%</entry></row><row><entry /><entry /><entry>C</entry><entry>92.9%</entry></row><row><entry /><entry /><entry>G</entry><entry> 2.8%</entry></row><row><entry /><entry /><entry>T</entry><entry> 3.9%</entry></row><row><entry /><entry>BE4-(GGS)₃</entry><entry>A</entry><entry> 1.3%</entry></row><row><entry /><entry /><entry>C</entry><entry> 9.9%</entry></row><row><entry /><entry /><entry>G</entry><entry> 7.9%</entry></row><row><entry /><entry /><entry>T</entry><entry>80.8%</entry></row><row><entry /><entry>BE4-XTEN</entry><entry>A</entry><entry> 2.3%</entry></row><row><entry /><entry /><entry>C</entry><entry>15.9%</entry></row><row><entry /><entry /><entry>G</entry><entry>12.2%</entry></row><row><entry /><entry /><entry>T</entry><entry>69.6%</entry></row><row><entry /><entry>BE4-32aa</entry><entry>A</entry><entry> 1.3%</entry></row><row><entry /><entry /><entry>C</entry><entry>14.9%</entry></row><row><entry /><entry /><entry>G</entry><entry> 9.9%</entry></row><row><entry /><entry /><entry>T</entry><entry>73.9%</entry></row><row><entry /><entry>BE4-2XUGI</entry><entry>A</entry><entry> 0.6%</entry></row><row><entry /><entry /><entry>C</entry><entry>23.4%</entry></row><row><entry /><entry /><entry>G</entry><entry> 3.8%</entry></row><row><entry /><entry /><entry>T</entry><entry>72.2%</entry></row><row><entry /><entry namest="offset" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0791<tables id="TABLE-US-00169" num="00169"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 13</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>HEK4</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="35pt" align="left" /><colspec colname="1" colwidth="91pt" align="left" /><colspec colname="2" colwidth="91pt" align="center" /><tbody valign="top"><row><entry /><entry>HEK4</entry><entry>C₅</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="offset" colwidth="35pt" align="left" /><colspec colname="1" colwidth="77pt" align="left" /><colspec colname="2" colwidth="14pt" align="center" /><colspec colname="3" colwidth="91pt" align="center" /><tbody valign="top"><row><entry /><entry>BE3</entry><entry>A</entry><entry>8.40%</entry></row><row><entry /><entry /><entry>C</entry><entry>41.89% </entry></row><row><entry /><entry /><entry>G</entry><entry>24.54% </entry></row><row><entry /><entry /><entry>T</entry><entry>25.17% </entry></row><row><entry /><entry>pmCDA1</entry><entry>A</entry><entry>0.50%</entry></row><row><entry /><entry /><entry>C</entry><entry>87.53% </entry></row><row><entry /><entry /><entry>G</entry><entry>0.01%</entry></row><row><entry /><entry /><entry>T</entry><entry>11.95% </entry></row><row><entry /><entry>hAID</entry><entry>A</entry><entry>0.93%</entry></row><row><entry /><entry /><entry>C</entry><entry>71.32% </entry></row><row><entry /><entry /><entry>G</entry><entry>0.69%</entry></row><row><entry /><entry /><entry>T</entry><entry>27.06% </entry></row><row><entry /><entry>hAPOBEC3G</entry><entry>A</entry><entry>0.12%</entry></row><row><entry /><entry /><entry>C</entry><entry>99.37% </entry></row><row><entry /><entry /><entry>G</entry><entry>0.35%</entry></row><row><entry /><entry /><entry>T</entry><entry>0.16%</entry></row><row><entry /><entry>BE4-N</entry><entry>A</entry><entry> 7.3%</entry></row><row><entry /><entry /><entry>C</entry><entry>56.6%</entry></row><row><entry /><entry /><entry>G</entry><entry>25.7%</entry></row><row><entry /><entry /><entry>T</entry><entry>10.3%</entry></row><row><entry /><entry>BE4-SSB</entry><entry>A</entry><entry> 2.1%</entry></row><row><entry /><entry /><entry>C</entry><entry>86.8%</entry></row><row><entry /><entry /><entry>G</entry><entry> 5.8%</entry></row><row><entry /><entry /><entry>T</entry><entry> 5.2%</entry></row><row><entry /><entry>BE4-(GGS)₃</entry><entry>A</entry><entry> 6.7%</entry></row><row><entry /><entry /><entry>C</entry><entry>13.0%</entry></row><row><entry /><entry /><entry>G</entry><entry>19.8%</entry></row><row><entry /><entry /><entry>T</entry><entry>60.5%</entry></row><row><entry /><entry>BE4-XTEN</entry><entry>A</entry><entry> 7.5%</entry></row><row><entry /><entry /><entry>C</entry><entry>19.7%</entry></row><row><entry /><entry /><entry>G</entry><entry>25.4%</entry></row><row><entry /><entry /><entry>T</entry><entry>47.4%</entry></row><row><entry /><entry>BE4-32aa</entry><entry>A</entry><entry> 7.9%</entry></row><row><entry /><entry /><entry>C</entry><entry>21.8%</entry></row><row><entry /><entry /><entry>G</entry><entry>25.1%</entry></row><row><entry /><entry /><entry>T</entry><entry>45.3%</entry></row><row><entry /><entry>BE4-2XUGI</entry><entry>A</entry><entry> 3.4%</entry></row><row><entry /><entry /><entry>C</entry><entry>22.2%</entry></row><row><entry /><entry /><entry>G</entry><entry>12.4%</entry></row><row><entry /><entry /><entry>T</entry><entry>62.0%</entry></row><row><entry /><entry namest="offset" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0792<tables id="TABLE-US-00170" num="00170"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 14</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>RNF2</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="35pt" align="left" /><colspec colname="1" colwidth="91pt" align="left" /><colspec colname="2" colwidth="91pt" align="center" /><tbody valign="top"><row><entry /><entry>RNF2</entry><entry>C₆</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="offset" colwidth="35pt" align="left" /><colspec colname="1" colwidth="77pt" align="left" /><colspec colname="2" colwidth="14pt" align="center" /><colspec colname="3" colwidth="91pt" align="center" /><tbody valign="top"><row><entry /><entry>BE3</entry><entry>A</entry><entry>2.46%</entry></row><row><entry /><entry /><entry>C</entry><entry>46.65% </entry></row><row><entry /><entry /><entry>G</entry><entry>19.87% </entry></row><row><entry /><entry /><entry>T</entry><entry>31.03% </entry></row><row><entry /><entry>pmCDA1</entry><entry>A</entry><entry>0.60%</entry></row><row><entry /><entry /><entry>C</entry><entry>83.52% </entry></row><row><entry /><entry /><entry>G</entry><entry>1.33%</entry></row><row><entry /><entry /><entry>T</entry><entry>14.55% </entry></row><row><entry /><entry>hAID</entry><entry>A</entry><entry>0.36%</entry></row><row><entry /><entry /><entry>C</entry><entry>75.03% </entry></row><row><entry /><entry /><entry>G</entry><entry>3.20%</entry></row><row><entry /><entry /><entry>T</entry><entry>21.40% </entry></row><row><entry /><entry>hAPOBEC3G</entry><entry>A</entry><entry>0.10%</entry></row><row><entry /><entry /><entry>C</entry><entry>86.60% </entry></row><row><entry /><entry /><entry>G</entry><entry>3.70%</entry></row><row><entry /><entry /><entry>T</entry><entry>9.59%</entry></row><row><entry /><entry>BE4-N</entry><entry>A</entry><entry> 5.1%</entry></row><row><entry /><entry /><entry>C</entry><entry>50.0%</entry></row><row><entry /><entry /><entry>G</entry><entry>28.8%</entry></row><row><entry /><entry /><entry>T</entry><entry>16.2%</entry></row><row><entry /><entry>BE4-SSB</entry><entry>A</entry><entry> 1.1%</entry></row><row><entry /><entry /><entry>C</entry><entry>89.9%</entry></row><row><entry /><entry /><entry>G</entry><entry> 4.9%</entry></row><row><entry /><entry /><entry>T</entry><entry> 4.1%</entry></row><row><entry /><entry>BE4-(GGS)₃</entry><entry>A</entry><entry> 2.0%</entry></row><row><entry /><entry /><entry>C</entry><entry>23.0%</entry></row><row><entry /><entry /><entry>G</entry><entry>14.0%</entry></row><row><entry /><entry /><entry>T</entry><entry>61.0%</entry></row><row><entry /><entry>BE4-XTEN</entry><entry>A</entry><entry> 2.6%</entry></row><row><entry /><entry /><entry>C</entry><entry>32.4%</entry></row><row><entry /><entry /><entry>G</entry><entry>16.0%</entry></row><row><entry /><entry /><entry>T</entry><entry>49.0%</entry></row><row><entry /><entry>BE4-32aa</entry><entry>A</entry><entry> 2.2%</entry></row><row><entry /><entry /><entry>C</entry><entry>29.2%</entry></row><row><entry /><entry /><entry>G</entry><entry>18.5%</entry></row><row><entry /><entry /><entry>T</entry><entry>50.0%</entry></row><row><entry /><entry>BE4-2XUGI</entry><entry>A</entry><entry> 0.7%</entry></row><row><entry /><entry /><entry>C</entry><entry>45.0%</entry></row><row><entry /><entry /><entry>G</entry><entry> 6.5%</entry></row><row><entry /><entry /><entry>T</entry><entry>47.8%</entry></row><row><entry /><entry namest="offset" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

Example 15: Base Editors Comprising a Cpf1 Nickase that Cleaves the Targeted Strand

0793As discussed above, nucleic acid programmable DNA binding proteins (napDNAbp) of any of the fusion proteins provided herein may be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). Cpf1 nickases, for example, a Cpf1 nickase (R1225A in AsCpf1; and R1138A in LbCpf1) that cleaves the non-target strand have been described in Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; the entire contents of which is hereby incorporated by reference. However, a nickase (e.g., a Cpf1 nickase of a base editor protein) that cleaves the target strand is expected to improve base editing efficiency.

0794A fluorescent labeled DNA was used to identify a Cpf1 mutant that preferentially nicks the target strand, rather than the non-target strand (see <figref idref="DRAWINGS">FIG. 116</figref>). In <figref idref="DRAWINGS">FIG. 116</figref>, the top strand of DNA constructs 1-3, is the non-target strand and the bottom strand is the target strand. An in vitro assay is carried out using wild-type LbCpf1, R836A (LbCpf1), and R1138A (LbCpf1). R836A (LbCpf1) appears to be a “crippled” nickase, meaning it cuts the target strand more efficiently than the non-target strand. As shown in <figref idref="DRAWINGS">FIG. 117</figref>, the non-target strand is uncut, no fluorescent 350 piece is observed. After two hours, both strands are cut. Differing intensities suggest more target strands are cut than non-target strands.

0000Establishing a Base Editing Window with AsCp1-BE3

0795Base editing proteins (e.g., BE3 (SpCas9-BE3)) having LbCpf1(R836A) or AsCpf1(R912A) as the napDNAbp were shown to edit bases at low efficiency (0.1% to 0.4%). A base editor with a AsCpf1 (R912A) napDNAbp more efficiently mutated a target C at EMX1, FANCF, HEK3 and HEK4 sites. The editing window of the constructs tested appears to be from the 7^thbase to the 11^thbase. The numbers are consistent with the trend with BE3 having highest numbers and self-defeating BE (i.e., APOBEC-AsCpf1(R1225A)-UGI, which cleaves the non-target strand) having lower ones. See <figref idref="DRAWINGS">FIG. 118</figref> Positive control with Cas9-BE3 on EMX1: 5-6%. Indel values for AsCpf1: >20%. R912 in AsCpf1 is conserved across many members of the Cpf1 family. The corresponding residue in LbCpf1 is R836, which is believed to be a “crippled” nickase when the R is mutated to an A.

0000Optimization of Cpf1-BE (Linkers)

0796Indel data suggests that Cpf1 can access DNA target sites. Thus, optimization of Cpf1 base editing proteins has focused on specific APOBEC proteins, linkers, and/or UGI domains. The construct shown in <figref idref="DRAWINGS">FIG. 119</figref> was tested, with varying linkers using both LbCpf1(R836A) and AsCpf1(R912A). In short, different linker sequences (i.e., XTEN, GGS, (GGS)₃(SEQ ID NO: 610), (GGS)₅(SEQ ID NO: 610), and (GGS)₇(SEQ ID NO: 610)) between the APOBEC and Cpf1 domain (e.g., AsCpf1 or LbCpf1) were tested. See <figref idref="DRAWINGS">FIG. 120</figref>. The constructs were tested for their ability to mutate the C₈residue of the HEK3 site, which is TGCTTCTC₈CAGCCCTGGCCTGG (SEQ ID NO: 592). Editing levels for base editing proteins with AsCpf1 reached to over 1%, while base editing proteins with LbCpf1 showed a comparative reduction in base editing efficiency. As shown in <figref idref="DRAWINGS">FIG. 121</figref>, linkers from a database maintained by the Centre of Integrative Bioinformatics VU did not show as significant an improvement as GGS-type linkers for AsCpf1-BE3. The linkers shown in <figref idref="DRAWINGS">FIG. 121</figref> are shown below:

0797<tables id="TABLE-US-00171" num="00171"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="35pt" align="left" /><colspec colname="2" colwidth="28pt" align="center" /><colspec colname="3" colwidth="154pt" align="left" /><thead><row><entry namest="1" nameend="3" align="center" rowsep="1" /></row><row><entry /><entry>Length</entry><entry /></row><row><entry>PDB_code</entry><entry>(aa)</entry><entry>Sequence</entry></row><row><entry namest="1" nameend="3" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="1" colwidth="35pt" align="left" /><colspec colname="2" colwidth="28pt" align="char" char="." /><colspec colname="3" colwidth="84pt" align="left" /><colspec colname="4" colwidth="70pt" align="left" /><tbody valign="top"><row><entry>1au7A_1</entry><entry>10</entry><entry>KRRTTISIAA</entry><entry>(SEQ ID NO: 593)</entry></row><row><entry></entry></row><row><entry>1c1kA_1</entry><entry>19</entry><entry>ALVFYREYIGRLKQIKFKF</entry><entry>(SEQ ID NO: 594)</entry></row><row><entry></entry></row><row><entry>1c20A_1</entry><entry>14</entry><entry>LPIMAKSVLDLYEL</entry><entry>(SEQ ID NO: 595)</entry></row><row><entry></entry></row><row><entry>1ee8A_1</entry><entry>5</entry><entry>LLRLG</entry><entry>(SEQ ID NO: 596)</entry></row><row><entry></entry></row><row><entry>1f1zA_1</entry><entry>15</entry><entry>TDKEINPVVKENIEW</entry><entry>(SEQ ID NO: 597)</entry></row><row><entry></entry></row><row><entry>1ignA 1</entry><entry>8</entry><entry>PPSIKRKF</entry><entry>(SEQ ID NO: 598)</entry></row><row><entry></entry></row><row><entry>1jmcA_1</entry><entry>9</entry><entry>LPTVQFDFT</entry><entry>(SEQ ID NO: 599)</entry></row><row><entry></entry></row><row><entry>1sfe_1</entry><entry>14</entry><entry>LPLDIRGTAFQQQV</entry><entry>(SEQ ID NO: 600)</entry></row><row><entry></entry></row><row><entry>2ezx_1</entry><entry>8</entry><entry>AYVVLGQF</entry><entry>(SEQ ID NO: 601)</entry></row><row><entry></entry></row><row><entry>2reb_1</entry><entry>8</entry><entry>INFYGELV</entry><entry>(SEQ ID NO: 602)</entry></row><row><entry namest="1" nameend="4" align="center" rowsep="1" /></row></tbody></tgroup></table></tables><br /> Optimization of Cpf1-BE (Orientations)

0798Cas9 has a stretch of amino acids between the C and N termini (see red square, <figref idref="DRAWINGS">FIG. 123</figref>) while AsCpf1 does not (see <figref idref="DRAWINGS">FIG. 122</figref>). Moreover, AsCpf1 has a shorter distance between the N and C termini. These observations indicate potential interference between APOBEC (on N terminus) and UGI (on C terminus) through which UGI may hinder APOBEC access to the non-target strand. One solution is to move APOBEC and UGI onto the same terminus, either N or C. Accordingly, constructs having the architecture NLS-UGI-APOBEC-XTEN-AsCpf1; UGI-APOBEC-XTEN-AsCpf1-NLS; and AsCpf1-XTEN-APOBEC-NLS will be tested.

0000Optimization of Cpf1-BE (Internal Truncation)

0799There is no known crystal structure of Cpf1 in which the non-target strand is resolved (see <figref idref="DRAWINGS">FIG. 124</figref>, cyan). It is believed that the editing window should lie within the red circle as shown in <figref idref="DRAWINGS">FIG. 124</figref>. There is a helical region (see square in <figref idref="DRAWINGS">FIG. 124</figref>) that may be obstructing APOBEC. This region comprises the amino acid sequence K(661)KTGDQK(667) (SEQ ID NO: 603).

0800To test the whether the removal of two, four or six residues improves base editing efficiency, experiments were conducted with a base editor having a AsCpf1(R912A) napDNAbp, using HEK3 as the target site. Editing levels increase to approximately 2.6%—a 6-fold increase from control levels when T663 and D665 are deleted (see Table 7, below). The construct used in this experiment was APOBEC-XTEN-AsCpf1(R912A)-SGGS-UGI

0801<tables id="TABLE-US-00172" num="00172"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="offset" colwidth="14pt" align="left" /><colspec colname="1" colwidth="91pt" align="left" /><colspec colname="2" colwidth="42pt" align="center" /><colspec colname="3" colwidth="70pt" align="center" /><thead><row><entry /><entry namest="offset" nameend="3" rowsep="1">TABLE 7</entry></row><row><entry /><entry namest="offset" nameend="3" align="center" rowsep="1" /></row><row><entry /><entry>Deletions</entry><entry>Editing at C8</entry><entry>Editing at C9</entry></row><row><entry /><entry namest="offset" nameend="3" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry /><entry>T663, D665</entry><entry>2.59%</entry><entry>1.29%</entry></row><row><entry /><entry>K662, T663, D665, Q666</entry><entry>0.15%</entry><entry>0.15%</entry></row><row><entry /><entry>K661, K662, T663, D665,</entry><entry>0.22%</entry><entry>0.21%</entry></row><row><entry /><entry>Q666, K667</entry></row><row><entry /><entry namest="offset" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

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Example 16: Improving DNA Specificity and Applicability of Base Editing Through Protein Engineering and Protein Delivery

0888Base editing, a genome editing approach that enables the programmable conversion of one base pair into another without double-stranded DNA cleavage, excess stochastic insertions and deletions, or dependence on homology-directed repair was developed. The application of base editing is limited by off-target activity and reliance on intracellular DNA delivery. Here two advances are described that address these limitations. First, off-target base editing has been reduced by installing mutations into the third-generation base editor (BE3) to generate a high-fidelity base editor (HF-BE3). Next, BE3 and HF-BE3 are purified and delivered as ribonucleoprotein (RNP) complexes into mammalian cells, establishing DNA-free base editing. RNP delivery of BE3 confers higher specificity even than plasmid transfection of HF-BE3, while maintaining comparable on-target editing levels. Finally, these advances are applied to deliver BE3 RNPs into both zebrafish embryos and the inner ear of live mice to achieve specific, DNA-free base editing in vivo.

0000Introduction

0889Traditional genome editing agents introduce double-stranded DNA breaks (DSBs) as the first step of genome editing^1-4. Cells respond to DSBs primarily through non-homologous end joining (NHEJ), resulting in stochastic insertions or deletions (indels) at the cleavage site^1,5. To generate more precise changes in genomic DNA, homology-directed repair (HDR) can be used to replace the genomic DNA surrounding the cleavage site with that of an exogenously supplied DNA donor template^6-8. Unfortunately, HDR is typically accompanied by an excess of indels resulting from competing NHEJ and is limited primarily to mitotic cells. In addition, most genome editing methods rely on delivery of exogenous plasmid or viral DNA into mammalian cells followed by intracellular expression of the agent^9-12. These delivery methods result in continuous, uncontrolled Cas9 and sgRNA expression even after the on-target locus has been edited, increasing the opportunity for genome editing at off-target loci^1,13.

0890Base editing, a different approach to genome editing that enables the direct, programmable, targeted conversion of a C:G base pair to a T:A base pair, was recently described^3,14. The third-generation base editor, BE3, contains in a single protein (i) a catalytically impaired Cas9 that opens a small single-stranded DNA bubble at a guide RNA-specified locus, (ii) a tethered single-strand-specific cytidine deaminase that converts C to U within a window of approximately five nucleotides in the single-stranded DNA bubble, (iii) a uracil glycosylase inhibitor (UGI) that inhibits base excision repair, thereby improving the efficiency and product selectivity of base editing, and (iv) nickase activity to manipulate cellular mismatch repair into replacing the G-containing DNA strand. The combination of these components enables efficient and permanent C to T (or G to A) conversion in mammalian cells with minimal indel formation. Since previously reported¹⁴, other researchers have confirmed the ability of this strategy and related approaches to facilitate Cas9-directed C to T conversion in mammalian cells^15-17and in plants¹⁸.

0891Here, two advances that greatly improve the DNA specificity of base editing and that allow base editing in vitro and in vivo without supplying exogenous DNA, which has been associated with a risk of recombination with the host genome and cytotoxicity, are described^18,19. First, a mutant form of BE3 incorporating mutations known to decrease the DNA affinity of Cas9²⁰that reduces off-target editing events with only a modest decrease in on-target editing activity is engineered. Next, it is revealed that lipid-mediated delivery of base editor proteins complexed with guide RNA results in even larger specificity enhancements with no apparent reduction in on-target base editing compared to plasmid DNA delivery. Delivery of base editors as RNPs typically reduces off-target editing to below measurable levels, even for a notoriously promiscuous guide RNA that targets a highly repetitive genomic DNA sequence, in cultured human and mouse cells. These advances enable highly specific, DNA-free in vivo base editing in mice and zebrafish to be demonstrated.

0000Results

0000Engineering a High-Fidelity Base Editor

0892Cas9 nucleases and their associated fusion constructs have been shown to bind and cleave DNA at off-target genomic loci^21-24. Joung and coworkers developed HF-Cas9, a high-fidelity SpCas9 variant containing four point mutations (N497A, R661A, Q695A, Q926A) that were designed to eliminate non-specific interactions between Cas9 and the phosphate backbone of the DNA target strand (<figref idref="DRAWINGS">FIG. 125A</figref>)²⁰consistent with the previous abrogation of non-specific DNA interactions in TALENs that greatly increased their DNA cleavage specificity²⁵. Since base editors operate on the non-target strand within the single-stranded DNA bubble created by Cas9¹⁴it can be hypothesized that introducing these four point mutations from HF-Cas9 into BE3 to generate “HF-BE3” might reduce off-target base editing without altering its base conversion capabilities (<figref idref="DRAWINGS">FIGS. 125A and 125C</figref>).

0893Plasmids encoding BE3 and HF-BE3 as His6-tagged proteins were overexpressed in <i>E. coli </i>and purified first by nickel affinity chromatography and then by cation exchange chromatography (<figref idref="DRAWINGS">FIGS. 126A-126B</figref>). Following extensive optimization of expression and purification conditions, BE3 and HF-BE3 protein can be routinely produced at a yield of ˜2 mg per liter of culture media (<figref idref="DRAWINGS">FIGS. 126A-126C</figref>).

0894The purified base editor proteins were used to compare base editing efficiency and the width of the editing window of HF-BE3 and BE3 biochemically. In vitro C to U conversion efficiencies were measured in a synthetic dsDNA 79-mer with a protospacer comprised of TC repeats. The target dsDNA (250 nM) was incubated with BE3:sgRNA or HF-BE3:sgRNA (2 μM) for 30 min at 37° C. After incubation, the edited DNA was amplified using a uracil-tolerant polymerase and sequenced by high-throughput DNA sequencing (HTS). Comparable editing efficiencies and activity window widths were observed for HF-BE3 and BE3 in vitro (FIG. <b>125</b>B). These findings indicate that introduction of the high-fidelity mutations into BE3 does not compromise inherent on-target base editing efficiency or change the width of the editing window of the resulting HF-BE3 protein in vitro.

0000HF-BE3 Enhances Editing Specificity Following DNA Transfection

0895Next, base editing efficiencies, specificities, and editing window widths of BE3 and HF-BE3 were compared in mammalian cells following plasmid DNA transfection. Four well-studied endogenous genomic loci (HEK293 site 3, FANCF, EMX1 and VEGFA site 2) were chosen to interrogate on- and off-target base editing in mammalian cells^14,24. VEGFA site 2 is highly repetitive, containing 14 Cs out of 20 protospacer nucleotides, and is associated with notoriously high rate of known off-target genome editing^20,22,24,26. This site was chosen to be included because it poses a formidable specificity challenge. In contrast with most nuclease-based genome editing applications, base editing relies on the precise location of the protospacer to place the target nucleotide within the editing window and usually little or no flexibility in the choice of guide RNA is available. Therefore, the development of base editors with enhanced specificities even for highly repetitive, promiscuous sgRNA targets is crucial^{3, 14}.

0896The on-target locus and known off-target loci were amplified by PCR and analyzed by HTS following plasmid transfection²⁴with each of the four base editor:sgRNA pairs. On-target editing in HEK293T cells for these four endogenous genomic loci was slightly reduced by introduction of the HF mutations; editing averaged 29±5% with BE3, and 21±3% (mean±s.e.m. for n=3 biological replicates) for HF-BE3 (<figref idref="DRAWINGS">FIGS. 127A-127D, 128A</figref>).

0897For each of the three standard, non-repetitive target sites (HEK293 site 3, FANCF, and EMX1), the three most frequently modified off-target loci that contain a C within the editing window from the off-target loci previously reported to be modified from treatment with Cas9 and the same guide RNA were examined (Table 15)²⁴. When cells were transfected with BE3 plasmid, C→T conversion across the nine most frequently modified Cas9 off-target loci for HEK293 site 3, FANCF, and EMX1 averaged 1.1±0.3% (<figref idref="DRAWINGS">FIGS. 127A-C</figref>; mean±s.d. for n=3 biological replicates). Installation of the HF mutations reduced the absolute level of mean off-target editing by 37-fold to 0.03±0.005%, with only one instance of measureable off-target C→T conversion (<figref idref="DRAWINGS">FIG. 127A</figref>; EMX1 C₅at off-target 1).

0898<tables id="TABLE-US-00173" num="00173"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="441pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 15</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Protospacer and PAM sequences for the on- and off-target human genomic loci studied</entry></row><row><entry>in this work. The off-target sites were chosen based on their GUIDE-Seq read count ⁴⁵. Cytosines</entry></row><row><entry>within the editing window for a particular sgRNA are numbered. The PAM sequence is shown in</entry></row><row><entry>bold. Protospacer bases in off-target loci that differ from their respective on-target loci have been</entry></row><row><entry>underlined. For genomic sequences interrogated in murine samples, see FIG. 132E.</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="1" colwidth="154pt" align="left" /><colspec colname="2" colwidth="168pt" align="left" /><colspec colname="3" colwidth="63pt" align="center" /><colspec colname="4" colwidth="56pt" align="center" /><tbody valign="top"><row><entry /><entry /><entry /><entry>GUIDE-Seq</entry></row><row><entry>Site</entry><entry>Sequence</entry><entry>SEQ ID NO</entry><entry>count</entry></row><row><entry namest="1" nameend="4" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="1" colwidth="154pt" align="left" /><colspec colname="2" colwidth="168pt" align="left" /><colspec colname="3" colwidth="63pt" align="center" /><colspec colname="4" colwidth="56pt" align="char" char="." /><tbody valign="top"><row><entry>EMX1 on-target</entry><entry>GAGTC₅C₆GAGCAGAAGAAGAA<b>GGG</b></entry><entry>480</entry><entry>4,521</entry></row><row><entry></entry></row><row><entry>EMX1 off-target 1</entry><entry>GAGTC₅<u style="single">TA</u>AGCAGAAGAAGAA<b>GAG</b></entry><entry>481</entry><entry>1,445</entry></row><row><entry></entry></row><row><entry>EMX1 off-target 2</entry><entry>GAG<u style="single">G</u>C₅C₆GAGCAGAAGAA<u style="single">AG</u>A<b>CGG</b></entry><entry>482</entry><entry>700</entry></row><row><entry></entry></row><row><entry>EMX1 off-target 3</entry><entry>GAGTC₅C₆<u style="single">T</u>AGCAG<u style="single">G</u>AGAAGAA<b>GAG</b></entry><entry>483</entry><entry>390</entry></row><row><entry></entry></row><row><entry>HEK293 site 3 on-target</entry><entry>GGCC₄C₅AGACTGAGCACGTGA<b>TGG</b></entry><entry>484</entry><entry>2,074</entry></row><row><entry></entry></row><row><entry>HEK293 site 3 off-target 1</entry><entry><u style="single">CA</u>CC₄C₅AGACTGAGCACGTG<u style="single">C</u><b>TGG</b></entry><entry>485</entry><entry>327</entry></row><row><entry></entry></row><row><entry>HEK293 site 3 off-target 2</entry><entry>G<u style="single">A</u>C<u style="single">A</u>C₅AGACTG<u style="single">G</u>GCACGTGA<b>GGG</b></entry><entry>486</entry><entry>306</entry></row><row><entry></entry></row><row><entry>HEK293 site 3 off-target 3</entry><entry><u style="single">A</u>GC<u style="single">T</u>C₅AGACTGAGCA<u style="single">A</u>GTGA<b>GGG</b></entry><entry>487</entry><entry>136</entry></row><row><entry></entry></row><row><entry>VEGFA site 2 on-target</entry><entry>GAC₃C₄C₅C₆C₇TC₉C₁₀ACCCCGCCTC<b>C</b></entry><entry>488</entry><entry>540</entry></row><row><entry /><entry><b>GG</b></entry><entry /><entry /></row><row><entry></entry></row><row><entry>VEGFA site 2 off-target 1</entry><entry><u style="single">CTA</u>C₄C₅C₆C₇TC₉C₁₀ACCCCGCCTC<b>CG</b></entry><entry>489</entry><entry>1,925</entry></row><row><entry /><entry><b>G</b></entry><entry /><entry /></row><row><entry></entry></row><row><entry>VEGFA site 2 off-target 2</entry><entry><chemistry id="CHEM-US-00262" num="00262"><img file="US11268082B2_D0262.tif" /></chemistry></entry><entry>490</entry><entry>1,549</entry></row><row><entry /><entry><b>G</b></entry><entry /><entry /></row><row><entry></entry></row><row><entry>VEGFA site 2 off-target 3</entry><entry><chemistry id="CHEM-US-00263" num="00263"><img file="US11268082B2_D0263.tif" /></chemistry></entry><entry>491</entry><entry>1,178</entry></row><row><entry /><entry><b>GG</b></entry><entry /><entry /></row><row><entry></entry></row><row><entry>VEGFA site 2 off-target 4</entry><entry><chemistry id="CHEM-US-00264" num="00264"><img file="US11268082B2_D0264.tif" /></chemistry></entry><entry>492</entry><entry>1,107</entry></row><row><entry /><entry><b>GG</b></entry><entry /><entry /></row><row><entry></entry></row><row><entry>FANCF on-target</entry><entry>GGAATC₆C₇C₈TTC₁₁TGCAGCACC<b>TGG</b></entry><entry>493</entry><entry>4,816</entry></row><row><entry></entry></row><row><entry>FANCF off-target 1</entry><entry><chemistry id="CHEM-US-00265" num="00265"><img file="US11268082B2_D0265.tif" /></chemistry></entry><entry>494</entry><entry>2,099</entry></row><row><entry /><entry><b>G</b></entry><entry /><entry /></row><row><entry></entry></row><row><entry>FANCF off-target 2</entry><entry><chemistry id="CHEM-US-00266" num="00266"><img file="US11268082B2_D0266.tif" /></chemistry></entry><entry>495</entry><entry>524</entry></row><row><entry /><entry><b>G</b></entry><entry /><entry /></row><row><entry></entry></row><row><entry>FANCF off-target 3</entry><entry><chemistry id="CHEM-US-00267" num="00267"><img file="US11268082B2_D0267.tif" /></chemistry></entry><entry>496</entry><entry>150</entry></row><row><entry /><entry><b>G</b></entry></row><row><entry namest="1" nameend="4" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0899To characterize HF-BE3 specificity on an extremely challenging site, BE3 and HF-BE3 off-target activity when targeting the highly repetitive VEGFA site 2 locus was compared. BE3 treatment lead to an average of 15±5% editing of cytosines located in the activity windows of the four tested off-target sites associated with this sgRNA (all average values quoted in this paragraph represent mean±s.d. for n 3 biological replicates). In contrast, HF-BE3 lead to a 3-fold reduction in absolute off-target editing (5.0±2.3%) at the same off-target sites (<figref idref="DRAWINGS">FIG. 127D</figref>). When compared to transfection of BE3, HF-BE3 significantly (p&lt;0.05, two-tailed Student's t test) reduced off-target editing at 27 of the 57 cytosines located at off-target loci (Table 16), while HF-BE3 treatment lead to a significant reduction (p&lt;0.05 two-tailed Student's t test) in on-target editing at only 3 of 16 the interrogated on-target cytosine residues.

0900<tables id="TABLE-US-00174" num="00174"><table frame="none" colsep="0" rowsep="0" pgwide="1"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="357pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 16</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>P-values for differences in base editing under different treatment conditions at all loci</entry></row><row><entry>evaluated in this study. p-values were calculated using the Student's two tailed t-test as described</entry></row><row><entry>in the Materials and Methods. When the p-value indicated a significant difference (p < 0.05), the</entry></row><row><entry>corresponding entry has been highlighted.</entry></row><row><entry>P values (Student's two-tailed t-test) for comparisons between listed treatments</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="11"><colspec colname="1" colwidth="35pt" align="left" /><colspec colname="2" colwidth="35pt" align="center" /><colspec colname="3" colwidth="35pt" align="center" /><colspec colname="4" colwidth="28pt" align="center" /><colspec colname="5" colwidth="28pt" align="center" /><colspec colname="6" colwidth="35pt" align="center" /><colspec colname="7" colwidth="28pt" align="center" /><colspec colname="8" colwidth="28pt" align="center" /><colspec colname="9" colwidth="35pt" align="center" /><colspec colname="10" colwidth="35pt" align="center" /><colspec colname="11" colwidth="35pt" align="center" /><tbody valign="top"><row><entry /><entry /><entry /><entry>plasmid</entry><entry /><entry /><entry>plasmid</entry><entry /><entry /><entry /><entry /></row><row><entry /><entry>plasmid</entry><entry /><entry>BE3</entry><entry /><entry>plasmid</entry><entry>HF-</entry></row><row><entry>Locus</entry><entry>BE3 vs</entry><entry>plasmid</entry><entry>vs</entry><entry>plasmid</entry><entry>HF-BE3</entry><entry>BE3 vs</entry><entry>plasmid</entry><entry>protein</entry><entry /><entry>protein</entry></row><row><entry>and</entry><entry>plasmid</entry><entry>BE3 vs</entry><entry>protein</entry><entry>BE3</entry><entry>vs</entry><entry>protein</entry><entry>HF-</entry><entry>BE3 vs</entry><entry>protein</entry><entry>HF-BE3</entry></row><row><entry>cytosine</entry><entry>HF-</entry><entry>protein</entry><entry>HF-</entry><entry>vs</entry><entry>protein</entry><entry>HF-</entry><entry>BE3 vs</entry><entry>protein</entry><entry>BE3 vs</entry><entry>vs</entry></row><row><entry>position</entry><entry>BE3</entry><entry>BE3</entry><entry>BE3</entry><entry>control</entry><entry>BE3</entry><entry>BE3</entry><entry>control</entry><entry>HF-BE3</entry><entry>control</entry><entry>control</entry></row><row><entry namest="1" nameend="11" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="11"><colspec colname="1" colwidth="35pt" align="left" /><colspec colname="2" colwidth="35pt" align="char" char="." /><colspec colname="3" colwidth="35pt" align="char" char="." /><colspec colname="4" colwidth="28pt" align="char" char="." /><colspec colname="5" colwidth="28pt" align="char" char="." /><colspec colname="6" colwidth="35pt" align="char" char="." /><colspec colname="7" colwidth="28pt" align="char" char="." /><colspec colname="8" colwidth="28pt" align="char" char="." /><colspec colname="9" colwidth="35pt" align="char" char="." /><colspec colname="10" colwidth="35pt" align="char" char="." /><colspec colname="11" colwidth="35pt" align="char" char="." /><tbody valign="top"><row><entry>EMX1,</entry><entry>0.053</entry><entry>0.000</entry><entry>0.001</entry><entry>0.000</entry><entry>0.416</entry><entry>0.056</entry><entry>0.003</entry><entry>0.023</entry><entry>0.000</entry><entry>0.000</entry></row><row><entry>C5</entry></row><row><entry>EMX1,</entry><entry>0.065</entry><entry>0.000</entry><entry>0.000</entry><entry>0.000</entry><entry>0.445</entry><entry>0.023</entry><entry>0.004</entry><entry>0.001</entry><entry>0.000</entry><entry>0.003</entry></row><row><entry>C6</entry></row><row><entry>FANCF,</entry><entry>0.152</entry><entry>0.017</entry><entry>0.003</entry><entry>0.000</entry><entry>0.137</entry><entry>0.003</entry><entry>0.000</entry><entry>0.002</entry><entry>0.000</entry><entry>0.004</entry></row><row><entry>C6</entry></row><row><entry>FANCF,</entry><entry>0.591</entry><entry>0.554</entry><entry>0.007</entry><entry>0.000</entry><entry>0.137</entry><entry>0.003</entry><entry>0.000</entry><entry>0.002</entry><entry>0.000</entry><entry>0.004</entry></row><row><entry>C7</entry></row><row><entry>FANCF,</entry><entry>0.011</entry><entry>0.026</entry><entry>0.004</entry><entry>0.000</entry><entry>0.958</entry><entry>0.018</entry><entry>0.000</entry><entry>0.023</entry><entry>0.000</entry><entry>0.007</entry></row><row><entry>C8</entry></row><row><entry>FANCF,</entry><entry>0.524</entry><entry>0.948</entry><entry>0.019</entry><entry>0.001</entry><entry>0.363</entry><entry>0.010</entry><entry>0.000</entry><entry>0.010</entry><entry>0.000</entry><entry>0.021</entry></row><row><entry>C11</entry></row><row><entry>HEK</entry><entry>0.061</entry><entry>0.001</entry><entry>0.071</entry><entry>0.000</entry><entry>0.002</entry><entry>0.002</entry><entry>0.005</entry><entry>0.00199</entry><entry>0.001</entry><entry>0.003</entry></row><row><entry>site 3,</entry></row><row><entry>C3</entry></row><row><entry>HEK</entry><entry>0.048</entry><entry>0.924</entry><entry>0.010</entry><entry>0.004</entry><entry>0.001</entry><entry>0.001</entry><entry>0.001</entry><entry>0.00004</entry><entry>0.000</entry><entry>0.001</entry></row><row><entry>site 3,</entry></row><row><entry>C4</entry></row><row><entry>HEK</entry><entry>0.291</entry><entry>0.592</entry><entry>0.016</entry><entry>0.006</entry><entry>0.243</entry><entry>0.243</entry><entry>0.002</entry><entry>0.00022</entry><entry>0.000</entry><entry>0.001</entry></row><row><entry>site 3,</entry></row><row><entry>C5</entry></row><row><entry>VEGFA</entry><entry>0.060</entry><entry>0.416</entry><entry>0.239</entry><entry>0.010</entry><entry>0.042</entry><entry>0.280</entry><entry>0.002</entry><entry>0.475</entry><entry>0.002</entry><entry>0.018</entry></row><row><entry>site 2,</entry></row><row><entry>C3</entry></row><row><entry>VEGFA</entry><entry>0.036</entry><entry>0.191</entry><entry>0.047</entry><entry>0.004</entry><entry>0.032</entry><entry>0.803</entry><entry>0.002</entry><entry>0.066</entry><entry>0.000</entry><entry>0.005</entry></row><row><entry>site 2,</entry></row><row><entry>C4</entry></row><row><entry>VEGFA</entry><entry>0.098</entry><entry>0.650</entry><entry>0.028</entry><entry>0.003</entry><entry>0.044</entry><entry>0.169</entry><entry>0.002</entry><entry>0.004</entry><entry>0.000</entry><entry>0.001</entry></row><row><entry>site 2,</entry></row><row><entry>C5</entry></row><row><entry>VEGFA</entry><entry>0.452</entry><entry>0.781</entry><entry>0.118</entry><entry>0.004</entry><entry>0.165</entry><entry>0.239</entry><entry>0.002</entry><entry>0.013</entry><entry>0.000</entry><entry>0.001</entry></row><row><entry>site 2,</entry></row><row><entry>C6</entry></row><row><entry>VEGFA</entry><entry>0.401</entry><entry>0.683</entry><entry>0.172</entry><entry>0.003</entry><entry>0.120</entry><entry>0.454</entry><entry>0.002</entry><entry>0.026</entry><entry>0.000</entry><entry>0.002</entry></row><row><entry>site 2,</entry></row><row><entry>C7</entry></row><row><entry>VEGFA</entry><entry>0.225</entry><entry>0.308</entry><entry>0.254</entry><entry>0.004</entry><entry>0.504</entry><entry>0.828</entry><entry>0.004</entry><entry>0.624</entry><entry>0.000</entry><entry>0.002</entry></row><row><entry>site 2,</entry></row><row><entry>C9</entry></row><row><entry>VEGFA</entry><entry>0.064</entry><entry>0.061</entry><entry>0.023</entry><entry>0.005</entry><entry>0.732</entry><entry>0.257</entry><entry>0.009</entry><entry>0.057</entry><entry>0.000</entry><entry>0.003</entry></row><row><entry>site 2,</entry></row><row><entry>C10</entry></row><row><entry>EMX1,</entry><entry>0.003</entry><entry>0.003</entry><entry>0.002</entry><entry>0.002</entry><entry>0.119</entry><entry>0.036</entry><entry>0.035</entry><entry>0.269</entry><entry>0.255</entry><entry>0.643</entry></row><row><entry>C5 off-</entry></row><row><entry>target 1</entry></row><row><entry>EMX1,</entry><entry>0.013</entry><entry>0.013</entry><entry>0.013</entry><entry>0.013</entry><entry>0.158</entry><entry>0.294</entry><entry>0.521</entry><entry>0.390</entry><entry>0.058</entry><entry>0.054</entry></row><row><entry>C5 off-</entry></row><row><entry>target 2</entry></row><row><entry>EMX1,</entry><entry>0.024</entry><entry>0.024</entry><entry>0.024</entry><entry>0.024</entry><entry>0.285</entry><entry>0.560</entry><entry>0.954</entry><entry>0.420</entry><entry>0.103</entry><entry>0.306</entry></row><row><entry>C6 off-</entry></row><row><entry>target 2</entry></row><row><entry>EMX1,</entry><entry>0.022</entry><entry>0.019</entry><entry>0.019</entry><entry>0.019</entry><entry>0.297</entry><entry>0.297</entry><entry>0.300</entry><entry>>0.99999</entry><entry>0.882</entry><entry>0.815</entry></row><row><entry>C5 off-</entry></row><row><entry>target 3</entry></row><row><entry>EMX1,</entry><entry>0.017</entry><entry>0.015</entry><entry>0.015</entry><entry>0.015</entry><entry>0.296</entry><entry>0.296</entry><entry>0.328</entry><entry>>0.99999</entry><entry>0.051</entry><entry>0.025</entry></row><row><entry>C6 off-</entry></row><row><entry>target 3</entry></row><row><entry>FANCF,</entry><entry>0.031</entry><entry>0.031</entry><entry>0.031</entry><entry>0.031</entry><entry>0.314</entry><entry>0.530</entry><entry>0.333</entry><entry>0.337</entry><entry>0.349</entry><entry>0.618</entry></row><row><entry>C5 off-</entry></row><row><entry>target 1</entry></row><row><entry>FANCF,</entry><entry>0.016</entry><entry>0.016</entry><entry>0.016</entry><entry>0.016</entry><entry>0.347</entry><entry>0.786</entry><entry>0.930</entry><entry>0.338</entry><entry>0.344</entry><entry>0.678</entry></row><row><entry>C6 off-</entry></row><row><entry>target 1</entry></row><row><entry>FANCF,</entry><entry>0.028</entry><entry>0.028</entry><entry>0.028</entry><entry>0.027</entry><entry>0.374</entry><entry>0.039</entry><entry>0.106</entry><entry>0.353</entry><entry>0.346</entry><entry>0.639</entry></row><row><entry>C7 off-</entry></row><row><entry>target 1</entry></row><row><entry>FANCF,</entry><entry>0.014</entry><entry>0.014</entry><entry>0.014</entry><entry>0.014</entry><entry>0.341</entry><entry>0.932</entry><entry>0.685</entry><entry>0.343</entry><entry>0.318</entry><entry>0.605</entry></row><row><entry>C8 off-</entry></row><row><entry>target 1</entry></row><row><entry>FANCF,</entry><entry>0.007</entry><entry>0.007</entry><entry>0.007</entry><entry>0.007</entry><entry>0.374</entry><entry>0.001</entry><entry>0.000</entry><entry>>0.99999</entry><entry>0.475</entry><entry>0.016</entry></row><row><entry>C11 off-</entry></row><row><entry>target 1</entry></row><row><entry>FANCF,</entry><entry>0.099</entry><entry>0.099</entry><entry>0.032</entry><entry>0.036</entry><entry>0.599</entry><entry>0.475</entry><entry>0.912</entry><entry>0.914</entry><entry>0.393</entry><entry>0.060</entry></row><row><entry>C6 off-</entry></row><row><entry>target 2</entry></row><row><entry>FANCF,</entry><entry>0.080</entry><entry>0.080</entry><entry>0.027</entry><entry>0.030</entry><entry>0.898</entry><entry>0.638</entry><entry>0.819</entry><entry>0.539</entry><entry>0.859</entry><entry>0.530</entry></row><row><entry>C7 off-</entry></row><row><entry>target 2</entry></row><row><entry>FANCF,</entry><entry>0.123</entry><entry>0.123</entry><entry>0.045</entry><entry>0.050</entry><entry>0.789</entry><entry>0.538</entry><entry>0.960</entry><entry>0.047</entry><entry>0.539</entry><entry>0.252</entry></row><row><entry>C8 off-</entry></row><row><entry>target 2</entry></row><row><entry>FANCF,</entry><entry>0.093</entry><entry>0.093</entry><entry>0.029</entry><entry>0.033</entry><entry>0.630</entry><entry>0.509</entry><entry>0.847</entry><entry>0.768</entry><entry>0.670</entry><entry>0.482</entry></row><row><entry>C10 off-</entry></row><row><entry>target 2</entry></row><row><entry>FANCF,</entry><entry>0.264</entry><entry>0.264</entry><entry>0.127</entry><entry>0.107</entry><entry>0.599</entry><entry>0.658</entry><entry>0.326</entry><entry>>0.99999</entry><entry>0.047</entry><entry>0.345</entry></row><row><entry>C11 off-</entry></row><row><entry>target 2</entry></row><row><entry>FANCF,</entry><entry>0.872</entry><entry>0.872</entry><entry>0.492</entry><entry>0.108</entry><entry>0.239</entry><entry>0.493</entry><entry>0.129</entry><entry>0.469</entry><entry>0.584</entry><entry>0.252</entry></row><row><entry>C6 off-</entry></row><row><entry>target 3</entry></row><row><entry>FANCF,</entry><entry>>0.99999</entry><entry>>0.99999</entry><entry>0.859</entry><entry>0.016</entry><entry>0.537</entry><entry>0.866</entry><entry>0.116</entry><entry>0.572</entry><entry>0.272</entry><entry>0.595</entry></row><row><entry>C7 off-</entry></row><row><entry>target 3</entry></row><row><entry>FANCF,</entry><entry>0.886</entry><entry>0.886</entry><entry>0.246</entry><entry>0.757</entry><entry>>0.99999</entry><entry>0.001</entry><entry>0.648</entry><entry>0.495</entry><entry>0.780</entry><entry>0.650</entry></row><row><entry>C8 off-</entry></row><row><entry>target 3</entry></row><row><entry>FANCF,</entry><entry>0.566</entry><entry>0.566</entry><entry>0.284</entry><entry>0.202</entry><entry>0.053</entry><entry>0.387</entry><entry>0.260</entry><entry>0.453</entry><entry>0.913</entry><entry>0.541</entry></row><row><entry>C10 off-</entry></row><row><entry>target 3</entry></row><row><entry>FANCF,</entry><entry>0.422</entry><entry>0.422</entry><entry>0.145</entry><entry>0.145</entry><entry>0.495</entry><entry>0.230</entry><entry>0.230</entry><entry>0.658</entry><entry>0.658</entry><entry>>0.99999</entry></row><row><entry>C11 off-</entry></row><row><entry>target 3</entry></row><row><entry>HEK293</entry><entry>>0.99999</entry><entry>0.910</entry><entry>0.412</entry><entry>0.326</entry><entry>0.910</entry><entry>0.412</entry><entry>0.326</entry><entry>0.293</entry><entry>0.223</entry><entry>0.480</entry></row><row><entry>site 3,</entry></row><row><entry>C3 off-</entry></row><row><entry>target 1</entry></row><row><entry>HEK293</entry><entry>>0.99999</entry><entry>0.994</entry><entry>0.437</entry><entry>0.391</entry><entry>0.994</entry><entry>0.437</entry><entry>0.391</entry><entry>0.495</entry><entry>0.451</entry><entry>0.614</entry></row><row><entry>site 3,</entry></row><row><entry>C4 off-</entry></row><row><entry>target 1</entry></row><row><entry>HEK293</entry><entry>>0.99999</entry><entry>0.616</entry><entry>0.814</entry><entry>0.337</entry><entry>0.616</entry><entry>0.814</entry><entry>0.337</entry><entry>0.459</entry><entry>0.116</entry><entry>0.481</entry></row><row><entry>site 3,</entry></row><row><entry>C5 off-</entry></row><row><entry>target 1</entry></row><row><entry>HEK293</entry><entry>0.285</entry><entry>0.473</entry><entry>0.100</entry><entry>0.473</entry><entry>0.141</entry><entry>0.735</entry><entry>0.141</entry><entry>0.038</entry><entry>>0.99999</entry><entry>0.038</entry></row><row><entry>site 3,</entry></row><row><entry>C3 off-</entry></row><row><entry>target 2</entry></row><row><entry>HEK293</entry><entry>0.375</entry><entry>0.294</entry><entry>0.177</entry><entry>0.294</entry><entry>0.687</entry><entry>0.428</entry><entry>0.687</entry><entry>0.064</entry><entry>>0.99999</entry><entry>0.064</entry></row><row><entry>site 3,</entry></row><row><entry>C5 off-</entry></row><row><entry>target 2</entry></row><row><entry>HEK293</entry><entry>0.053</entry><entry>0.624</entry><entry>0.374</entry><entry>0.624</entry><entry>0.554</entry><entry>0.154</entry><entry>0.554</entry><entry>0.872</entry><entry>>0.99999</entry><entry>0.872</entry></row><row><entry>site 3,</entry></row><row><entry>C9 off-</entry></row><row><entry>target 2</entry></row><row><entry>HEK293</entry><entry>0.067</entry><entry>0.116</entry><entry>0.768</entry><entry>0.435</entry><entry>0.519</entry><entry>0.230</entry><entry>0.561</entry><entry>0.349</entry><entry>0.768</entry><entry>0.643</entry></row><row><entry>site 3,</entry></row><row><entry>C3 off-</entry></row><row><entry>target 3</entry></row><row><entry>HEK293</entry><entry>0.011</entry><entry>0.011</entry><entry>0.011</entry><entry>0.011</entry><entry>0.016</entry><entry>0.643</entry><entry>0.435</entry><entry>0.184</entry><entry>0.025</entry><entry>0.346</entry></row><row><entry>site 3,</entry></row><row><entry>C5 off-</entry></row><row><entry>target 3</entry></row><row><entry>HEK293</entry><entry>>0.99999</entry><entry>0.374</entry><entry>0.652</entry><entry>0.811</entry><entry>0.132</entry><entry>0.539</entry><entry>0.776</entry><entry>0.609</entry><entry>0.643</entry><entry>0.893</entry></row><row><entry>site 3,</entry></row><row><entry>C9 off-</entry></row><row><entry>target 3</entry></row><row><entry>VEGFA</entry><entry>0.101</entry><entry>0.015</entry><entry>0.014</entry><entry>0.012</entry><entry>0.041</entry><entry>0.032</entry><entry>0.025</entry><entry>0.117</entry><entry>0.012</entry><entry>0.001</entry></row><row><entry>site 2,</entry></row><row><entry>C4 off-</entry></row><row><entry>target 1</entry></row><row><entry>VEGFA</entry><entry>0.060</entry><entry>0.013</entry><entry>0.012</entry><entry>0.010</entry><entry>0.078</entry><entry>0.062</entry><entry>0.044</entry><entry>0.201</entry><entry>0.009</entry><entry>0.012</entry></row><row><entry>site 2,</entry></row><row><entry>C5 off-</entry></row><row><entry>target 1</entry></row><row><entry>VEGFA</entry><entry>0.019</entry><entry>0.005</entry><entry>0.005</entry><entry>0.004</entry><entry>0.080</entry><entry>0.062</entry><entry>0.045</entry><entry>0.087</entry><entry>0.002</entry><entry>0.012</entry></row><row><entry>site 2,</entry></row><row><entry>C6 off-</entry></row><row><entry>target 1</entry></row><row><entry>VEGFA</entry><entry>0.017</entry><entry>0.004</entry><entry>0.004</entry><entry>0.003</entry><entry>0.080</entry><entry>0.060</entry><entry>0.037</entry><entry>0.076</entry><entry>0.001</entry><entry>0.002</entry></row><row><entry>site 2,</entry></row><row><entry>C7 off-</entry></row><row><entry>target 1</entry></row><row><entry>VEGFA</entry><entry>0.230</entry><entry>0.088</entry><entry>0.037</entry><entry>0.011</entry><entry>0.667</entry><entry>0.256</entry><entry>0.051</entry><entry>0.134</entry><entry>0.004</entry><entry>0.007</entry></row><row><entry>site 2,</entry></row><row><entry>C9 off-</entry></row><row><entry>target 1</entry></row><row><entry>VEGFA</entry><entry>0.535</entry><entry>0.136</entry><entry>0.103</entry><entry>0.035</entry><entry>0.283</entry><entry>0.211</entry><entry>0.050</entry><entry>0.717</entry><entry>0.028</entry><entry>0.010</entry></row><row><entry>site 2,</entry></row><row><entry>C10 off-</entry></row><row><entry>target 1</entry></row><row><entry>VEGFA</entry><entry>0.038</entry><entry>0.004</entry><entry>0.003</entry><entry>0.003</entry><entry>0.087</entry><entry>0.051</entry><entry>0.048</entry><entry>0.063</entry><entry>0.048</entry><entry>0.134</entry></row><row><entry>site 2,</entry></row><row><entry>C4 off-</entry></row><row><entry>target 2</entry></row><row><entry>VEGFA</entry><entry>0.033</entry><entry>0.004</entry><entry>0.004</entry><entry>0.004</entry><entry>0.078</entry><entry>0.061</entry><entry>0.059</entry><entry>0.028</entry><entry>0.020</entry><entry>0.248</entry></row><row><entry>site 2,</entry></row><row><entry>C5 off-</entry></row><row><entry>target 2</entry></row><row><entry>VEGFA</entry><entry>0.026</entry><entry>0.005</entry><entry>0.005</entry><entry>0.004</entry><entry>0.051</entry><entry>0.038</entry><entry>0.038</entry><entry>0.043</entry><entry>0.038</entry><entry>0.783</entry></row><row><entry>site 2,</entry></row><row><entry>C6 off-</entry></row><row><entry>target 2</entry></row><row><entry>VEGFA</entry><entry>0.053</entry><entry>0.006</entry><entry>0.005</entry><entry>0.005</entry><entry>0.072</entry><entry>0.056</entry><entry>0.055</entry><entry>0.078</entry><entry>0.064</entry><entry>0.704</entry></row><row><entry>site 2,</entry></row><row><entry>C7 off-</entry></row><row><entry>target 2</entry></row><row><entry>VEGFA</entry><entry>0.071</entry><entry>0.006</entry><entry>0.006</entry><entry>0.006</entry><entry>0.079</entry><entry>0.065</entry><entry>0.065</entry><entry>0.118</entry><entry>0.107</entry><entry>0.703</entry></row><row><entry>site 2,</entry></row><row><entry>C8 off-</entry></row><row><entry>target 2</entry></row><row><entry>VEGFA</entry><entry>0.193</entry><entry>0.008</entry><entry>0.007</entry><entry>0.006</entry><entry>0.103</entry><entry>0.090</entry><entry>0.084</entry><entry>0.068</entry><entry>0.007</entry><entry>0.217</entry></row><row><entry>site 2,</entry></row><row><entry>C9 off-</entry></row><row><entry>target 2</entry></row><row><entry>VEGFA</entry><entry>0.063</entry><entry>0.003</entry><entry>0.003</entry><entry>0.002</entry><entry>0.116</entry><entry>0.107</entry><entry>0.090</entry><entry>0.545</entry><entry>0.016</entry><entry>0.346</entry></row><row><entry>site 2,</entry></row><row><entry>C10 off-</entry></row><row><entry>target 2</entry></row><row><entry>VEGFA</entry><entry>0.005</entry><entry>0.003</entry><entry>0.003</entry><entry>0.003</entry><entry>0.091</entry><entry>0.031</entry><entry>0.030</entry><entry>0.116</entry><entry>0.107</entry><entry>0.158</entry></row><row><entry>site 2,</entry></row><row><entry>C4 off-</entry></row><row><entry>target 3</entry></row><row><entry>VEGFA</entry><entry>0.011</entry><entry>0.007</entry><entry>0.005</entry><entry>0.005</entry><entry>0.211</entry><entry>0.048</entry><entry>0.042</entry><entry>0.220</entry><entry>0.177</entry><entry>0.001</entry></row><row><entry>site 2,</entry></row><row><entry>C5 off-</entry></row><row><entry>target 3</entry></row><row><entry>VEGFA</entry><entry>0.020</entry><entry>0.005</entry><entry>0.003</entry><entry>0.003</entry><entry>0.142</entry><entry>0.038</entry><entry>0.033</entry><entry>0.193</entry><entry>0.149</entry><entry>0.015</entry></row><row><entry>site 2,</entry></row><row><entry>C6 off-</entry></row><row><entry>target 3</entry></row><row><entry>VEGFA</entry><entry>0.045</entry><entry>0.006</entry><entry>0.003</entry><entry>0.003</entry><entry>0.101</entry><entry>0.035</entry><entry>0.030</entry><entry>0.093</entry><entry>0.060</entry><entry>0.083</entry></row><row><entry>site 2,</entry></row><row><entry>C7 off-</entry></row><row><entry>target 3</entry></row><row><entry>VEGFA</entry><entry>0.069</entry><entry>0.007</entry><entry>0.005</entry><entry>0.005</entry><entry>0.087</entry><entry>0.045</entry><entry>0.039</entry><entry>0.120</entry><entry>0.067</entry><entry>0.041</entry></row><row><entry>site 2,</entry></row><row><entry>C8 off-</entry></row><row><entry>target 3</entry></row><row><entry>VEGFA</entry><entry>0.093</entry><entry>0.006</entry><entry>0.005</entry><entry>0.005</entry><entry>0.041</entry><entry>0.032</entry><entry>0.028</entry><entry>0.396</entry><entry>0.195</entry><entry>0.005</entry></row><row><entry>site 2,</entry></row><row><entry>C9 off-</entry></row><row><entry>target 3</entry></row><row><entry>VEGFA</entry><entry>0.342</entry><entry>0.011</entry><entry>0.008</entry><entry>0.007</entry><entry>0.109</entry><entry>0.081</entry><entry>0.069</entry><entry>0.273</entry><entry>0.098</entry><entry>0.036</entry></row><row><entry>site 2,</entry></row><row><entry>C10 off-</entry></row><row><entry>target 3</entry></row><row><entry>VEGFA</entry><entry>0.001</entry><entry>0.001</entry><entry>0.001</entry><entry>0.001</entry><entry>0.374</entry><entry>0.374</entry><entry>0.230</entry><entry>0.271</entry><entry>0.358</entry><entry>0.633</entry></row><row><entry>site 2,</entry></row><row><entry>C3 off-</entry></row><row><entry>target 4</entry></row><row><entry>VEGFA</entry><entry>0.007</entry><entry>0.006</entry><entry>0.006</entry><entry>0.006</entry><entry>0.137</entry><entry>0.137</entry><entry>0.137</entry><entry>0.592</entry><entry>0.862</entry><entry>0.690</entry></row><row><entry>site 2,</entry></row><row><entry>C4 off-</entry></row><row><entry>target 4</entry></row><row><entry>VEGFA</entry><entry>0.007</entry><entry>0.007</entry><entry>0.007</entry><entry>0.007</entry><entry>0.026</entry><entry>0.017</entry><entry>0.018</entry><entry>0.461</entry><entry>0.655</entry><entry>0.279</entry></row><row><entry>site 2,</entry></row><row><entry>C5 off-</entry></row><row><entry>target 4</entry></row><row><entry>VEGFA</entry><entry>0.005</entry><entry>0.004</entry><entry>0.004</entry><entry>0.004</entry><entry>0.021</entry><entry>0.018</entry><entry>0.018</entry><entry>0.398</entry><entry>0.546</entry><entry>0.149</entry></row><row><entry>site 2,</entry></row><row><entry>C6 off-</entry></row><row><entry>target 4</entry></row><row><entry>VEGFA</entry><entry>0.007</entry><entry>0.006</entry><entry>0.006</entry><entry>0.006</entry><entry>0.051</entry><entry>0.048</entry><entry>0.050</entry><entry>0.373</entry><entry>0.720</entry><entry>0.029</entry></row><row><entry>site 2,</entry></row><row><entry>C7 off-</entry></row><row><entry>target 4</entry></row><row><entry>VEGFA</entry><entry>0.007</entry><entry>0.006</entry><entry>0.006</entry><entry>0.006</entry><entry>0.092</entry><entry>0.092</entry><entry>0.092</entry><entry>0.325</entry><entry>0.014</entry><entry>0.275</entry></row><row><entry>site 2,</entry></row><row><entry>C8 off-</entry></row><row><entry>target 4</entry></row><row><entry>VEGFA</entry><entry>0.016</entry><entry>0.007</entry><entry>0.007</entry><entry>0.007</entry><entry>0.150</entry><entry>0.150</entry><entry>0.150</entry><entry>0.502</entry><entry>1.000</entry><entry>0.615</entry></row><row><entry>site 2,</entry></row><row><entry>C9 off-</entry></row><row><entry>target 4</entry></row><row><entry>VEGFA</entry><entry>0.213</entry><entry>0.009</entry><entry>0.009</entry><entry>0.009</entry><entry>0.261</entry><entry>0.261</entry><entry>0.261</entry><entry>0.653</entry><entry>0.575</entry><entry>0.660</entry></row><row><entry>site 2,</entry></row><row><entry>C10 off-</entry></row><row><entry>target 4</entry></row><row><entry namest="1" nameend="11" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0901In addition to considering the differences between absolute editing at off-target loci, the on-target:off-target editing specificity ratio was also calculated by dividing the observed on-target efficiency by the off-target efficiency (<figref idref="DRAWINGS">FIGS. 129A-129B</figref>). This metric takes into account any reduction in on-target editing associated with installation of the HF-mutations, and is useful for applications sensitive to both the efficiency and specificity of base editing. Off-target editing by HF-BE3 was below the detection limit of high-throughput sequencing for several off-target loci. For these cases, a conservative off-target editing efficiency equal to the upper limit of detection was assumed (0.025% C→T conversion; see Methods). Based on this analysis, the average improvement in specificity ratio upon installation of the HF mutations across all 34 target cytosines examined herein was 19-fold, when plasmid delivery of the two constructs was performed. These results collectively establish that for non-repetitive sites (<figref idref="DRAWINGS">FIG. 129A</figref>) as well as a highly repetitive site (<figref idref="DRAWINGS">FIG. 129B</figref>), HF-BE3 results in substantially enhanced base editing specificity with only a modest reduction in on-target editing efficiency compared to BE3.

0000RNP Delivery of BE3 Enables DNA-Free Base Editing

0902Next, the ability of BE3 in DNA-free, RNP form to mediate base editing when directly delivered into cultured human cells was studied. It has recently been established that cationic lipid reagents can potently deliver negatively charged proteins or protein:nucleic acid complexes into mammalian cells including ribonucleoprotein (RNP) complexes and that RNP delivery can substantially reduce off-target genome editing^27-29

0903The commercially available cationic lipid LIPOFECTAMINE® 2000 transfection reagent was combined with either purified BE3 protein or HF-BE3 protein after pre-complexation with a guide RNA targeting the EMX1, HEK293 site 3, FANCF, or VEGFA site 2 locus and the resulting lipid:RNP complexes were incubated with HEK293T cells. After 72 h, genomic DNA was harvested and on-target and off-target base editing was analyzed by high-throughput DNA sequencing. As with all Cas9-based technologies, substantial variations were observed in editing efficiency at different genomic loci (<figref idref="DRAWINGS">FIGS. 127 and 130</figref>). To display trends associated with in on-target editing efficiency between different treatments, mean on-target base editing efficiencies were calculated at the four tested loci (<figref idref="DRAWINGS">FIG. 128A</figref>). Protein delivery of BE3 (200 nM) lead to on-target editing efficiencies comparable to those observed with plasmid transfection (26±4% vs. 29±5% respectively; mean±s.e.m. for n=3 biological replicates; <figref idref="DRAWINGS">FIG. 128A</figref>).

0904In contrast, protein delivery of HF-BE3 reduced on-target editing compared to protein delivery of BE3 at the four genomic loci studied (average editing efficiency of 13±3% vs. 26±4%, respectively; mean±s.e.m. for n=3 biological replicates; <figref idref="DRAWINGS">FIG. 128A</figref>). Since HF-BE3 and BE3 have comparable editing efficiencies in a test tube (<figref idref="DRAWINGS">FIG. 125B</figref>) and editing is only slightly reduced when HF-BE3 is expressed from plasmids in HEK293T cells (<figref idref="DRAWINGS">FIG. 127A-D</figref>), it is tempting to speculate that the decreased efficiency of editing from HF-BE3 protein delivery may be a result of decreased HF-BE3 stability in mammalian cells. Lower stability could be offset by continual expression from a plasmid, but not following one-time protein delivery. This observation is consistent with a recent report of reduced on-target indel formation with purified HF-Cas9 compared to purified Cas9 when nucleofected into CD34⁺ hematopoietic stem and progenitor cells³⁰. While this work was in review, Kim et al demonstrated RNP delivery of BE3 into mouse embryos using electroporation³¹. To the best of the inventors' knowledge, the present approach is the first DNA-free technique capable of generating precise changes to individual nucleotides in mammalian cells without electroporation, which has limited in vivo therapeutic relevance.

0000RNP Delivery of Base Editors Greatly Enhances DNA Specificity

0905Importantly, while RNP delivery of BE3 and HF-BE3 led to substantial on-target base editing, no instances of measurable base editing (&lt;0.025%) were observed at any of the nine tested off-target loci associated with EMX1, FANCF and HEK293 site 3, (<figref idref="DRAWINGS">FIGS. 130A-130C</figref>). In contrast, plasmid delivery of BE3 lead to an average of 1.1±0.3% (mean±s.d. for n=3 biological replicates) off-editing across all sequenced cytosines within the base editing activity window, and detectable off-target editing at 11 of 16 off-target cytosines located at these nine off-target loci (<figref idref="DRAWINGS">FIGS. 127A-127D</figref>). At off-target loci of the three non-repetitive loci tested, BE3 protein delivery lead to a 26-fold higher average specificity ratio than that of plasmid delivery (<figref idref="DRAWINGS">FIG. 127A</figref>). These results reveal that RNP delivery of base editors dramatically increases the DNA specificity of base editing.

0906Protein delivery of either BE3 or HF-BE3 also resulted in greatly improved base editing specificity at the highly promiscuous VEGFA site 2 locus compared to plasmid delivery of either BE3 or HF-BE3 (compare <figref idref="DRAWINGS">FIGS. 127 and 130</figref>; see Table 16). Absolute frequencies of base editing at the off-target loci associated with this site were reduced upon protein delivery at least 10-fold for both BE3 (plasmid delivery: 15±4% off-target editing; protein delivery: 1.3±0.4% off-target editing; all values in this paragraph represent mean±s.d. for n=3 biological replicates) and HF-BE3 (plasmid delivery: 5±2% off-target editing; protein delivery: 0.5±0.1% off-target editing). Across all four studied loci, base editing specificity ratios for on-target:off-target editing increased an average of 66-fold for protein delivery of BE3 compared with plasmid delivery of BE3 (<figref idref="DRAWINGS">FIG. 129</figref>). Collectively, these results reveal that for both repetitive and non-repetitive target sites, RNP versus DNA delivery is a stronger determinant of base editing specificity than the presence or absence of the high-fidelity Cas9 mutations.

0907Neither introduction of the HF mutations nor delivery method substantially altered the low indel rates associated with base editing. Indel frequencies at all on-target loci across all treatment conditions in this study remained low (typically ≤5%; <figref idref="DRAWINGS">FIG. 131A</figref>), and the editing:indel ratio remained higher in all cases tested (typically ≥10-fold; <figref idref="DRAWINGS">FIG. 131B</figref>) than in previous studies using optimized HDR protocols^30,32,33. For non-repetitive sgRNAs, very few indels were observed at off-target loci (<figref idref="DRAWINGS">FIG. 131C</figref>), although it is noted that plasmid delivery of BE3 generated up to 5% indels for off-target loci associated with VEGFA site 2 (<figref idref="DRAWINGS">FIG. 131C</figref>).

0908Taken together, these results establish that protein delivery of base editors maintains on-target base editing efficiency and greatly enhances editing specificity relative to delivery of plasmid DNA.

0000RNP Delivery Decouples On- and Off-Target Editing

0909Given the striking enhancement of base editing specificity associated with protein delivery of BE3, it was investigated if this improvement was a result of a reduction in the total quantity of active genome editing agent delivered into the cell. Using the sgRNA targeting EMX1, a dose response study for plasmid (<figref idref="DRAWINGS">FIG. 128B</figref>) and protein delivery (<figref idref="DRAWINGS">FIG. 128C</figref>) was performed. To maximize transfection efficiency between treatment conditions, the volume of LIPOFECTAMINE® 2000 transfection reagent was 1.5 μL for all tests, and the base editor protein:sgRNA molar ratio was maintained at 1:1.1 for protein delivery. For plasmid delivery, a mass ratio of sgRNA plasmid:BE3 plasmid of 1:3 (molar ratio ˜1:1) and 1.5 μL of LIPOFECTAMINE® 2000 transfection reagent were used. Off-target base editing was observed under all conditions tested for plasmid delivery (<figref idref="DRAWINGS">FIG. 128B</figref>), but virtually no off-target editing under all protein delivery conditions tested (<figref idref="DRAWINGS">FIG. 128C</figref>).

0910Linear regression analysis was performed to assess the relationship between on- and off-target editing for plasmid and protein delivery. For plasmid delivery, off-target editing was closely associated with on-target editing rates (R²=0.95, p=0.0012 for non-zero slope, F-test), whereas there was no significant association between off-target and on-target editing using protein delivery (R²=0.078, p=0.59 for non-zero slope, F-test).

0911These data indicate that protein delivery of base editors offers an inherent specificity advantage that is independent of dosage. Together with the previous observations^29,34, these findings support a model in which the higher DNA specificity of base editing from protein delivery compared to DNA delivery arises from the ability of protein delivery to avoid extended exposure of the genome to base editors, thereby minimizing the opportunity of base editors to process off-target loci after on-target loci have already been modified.

0000DNA-Free Base Editing in Zebrafish and Mice

0912The above observations suggested the promise of protein delivery of BE3 to maintain on-target base editing while eliminating detectable off-target base editing. It was therefore tested whether protein delivery of BE3 could be used to generate specific point mutations in zebrafish by injecting BE3:sgRNA complexes targeting the tyrosinase locus into fertilized zebrafish embryos. Genomic DNA was harvested from the resultant zebrafish larvae 4 days post-injection and measured base editing and indel frequencies by high-throughput sequencing (<figref idref="DRAWINGS">FIG. 132A</figref>). Two of the three BE3:sgRNA complexes tested induced substantial point mutations in vivo (TYR1: C₃→T₃5.3±1.8%, TYR2: C₄→T₄4.3±2.1%; mean±s.d. of n=3 injected embryos; <figref idref="DRAWINGS">FIG. 132A</figref>). Sequences of zebrafish loci are listed in Table 17.

0913<tables id="TABLE-US-00175" num="00175"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 17</entry></row><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Number of HTS reads that align to the reference sequence</entry></row><row><entry>and pass the quality filters described in Materials and Methods.</entry></row><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="63pt" align="left" /><colspec colname="1" colwidth="84pt" align="center" /><colspec colname="2" colwidth="70pt" align="center" /><tbody valign="top"><row><entry /><entry>Replicate</entry><entry /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="5"><colspec colname="1" colwidth="63pt" align="left" /><colspec colname="2" colwidth="28pt" align="center" /><colspec colname="3" colwidth="28pt" align="center" /><colspec colname="4" colwidth="28pt" align="center" /><colspec colname="5" colwidth="70pt" align="left" /><tbody valign="top"><row><entry>Sample Description</entry><entry>1</entry><entry>2</entry><entry>3</entry><entry>Amplicon</entry></row><row><entry namest="1" nameend="5" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><tbody valign="top"><row><entry>HEK cell samples</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="5"><colspec colname="1" colwidth="63pt" align="left" /><colspec colname="2" colwidth="28pt" align="char" char="." /><colspec colname="3" colwidth="28pt" align="char" char="." /><colspec colname="4" colwidth="28pt" align="char" char="." /><colspec colname="5" colwidth="70pt" align="left" /><tbody valign="top"><row><entry>Protein, BE3</entry><entry>237256</entry><entry>295609</entry><entry>159391</entry><entry>VEGFA On Target</entry></row><row><entry>Protein, HF-BE3</entry><entry>383480</entry><entry>389874</entry><entry>383467</entry><entry>VEGFA On Target</entry></row><row><entry>Plasmid, BE3</entry><entry>315213</entry><entry>280891</entry><entry>335668</entry><entry>VEGFA On Target</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>196323</entry><entry>251965</entry><entry>369201</entry><entry>VEGFA On Target</entry></row><row><entry>Control</entry><entry>390748</entry><entry>395523</entry><entry>353614</entry><entry>VEGFA On Target</entry></row><row><entry>Protein, BE3</entry><entry>19280</entry><entry>26472</entry><entry>24799</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 1</entry></row><row><entry>Protein, HF-BE3</entry><entry>36383</entry><entry>30007</entry><entry>39193</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 1</entry></row><row><entry>Plasmid, BE3</entry><entry>35580</entry><entry>29557</entry><entry>22243</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 1</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>40371</entry><entry>27187</entry><entry>28248</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 1</entry></row><row><entry>Control</entry><entry>35106</entry><entry>37274</entry><entry>34939</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 1</entry></row><row><entry>Protein, BE3</entry><entry>82978</entry><entry>124689</entry><entry>83840</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 1</entry></row><row><entry>Protein, HF-BE3</entry><entry>142404</entry><entry>140482</entry><entry>142220</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 1</entry></row><row><entry>Plasmid, BE3</entry><entry>112027</entry><entry>117071</entry><entry>100894</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 1</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>114187</entry><entry>98876</entry><entry>122553</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 1</entry></row><row><entry>Control</entry><entry>76854</entry><entry>90547</entry><entry>89271</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 1</entry></row><row><entry>Protein, BE3</entry><entry>14514</entry><entry>25515</entry><entry>19325</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 2</entry></row><row><entry>Protein, HF-BE3</entry><entry>24678</entry><entry>24363</entry><entry>25312</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 2</entry></row><row><entry>Plasmid, BE3</entry><entry>16945</entry><entry>19918</entry><entry>10225</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 2</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>12200</entry><entry>14769</entry><entry>17797</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 2</entry></row><row><entry>Control</entry><entry>8739</entry><entry>13648</entry><entry>7818</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 2</entry></row><row><entry>Protein, BE3</entry><entry>17924</entry><entry>111693</entry><entry>173909</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 3</entry></row><row><entry>Protein, HF-BE3</entry><entry>243899</entry><entry>300503</entry><entry>276139</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 3</entry></row><row><entry>Plasmid, BE3</entry><entry>208476</entry><entry>291370</entry><entry>155430</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 3</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>117174</entry><entry>154033</entry><entry>199152</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 3</entry></row><row><entry>Control</entry><entry>119263</entry><entry>170436</entry><entry>121686</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 3</entry></row><row><entry>Protein, BE3</entry><entry>237799</entry><entry>262947</entry><entry>185371</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 4</entry></row><row><entry>Protein, HF-BE3</entry><entry>313253</entry><entry>233699</entry><entry>244922</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 4</entry></row><row><entry>Plasmid, BE3</entry><entry>243094</entry><entry>230316</entry><entry>234421</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 4</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>170958</entry><entry>160091</entry><entry>140693</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 4</entry></row><row><entry>Control</entry><entry>158691</entry><entry>148720</entry><entry>137270</entry><entry>VEGFA Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 4</entry></row><row><entry>Protein, BE3</entry><entry>28684</entry><entry>28237</entry><entry>43315</entry><entry>HEK3 On Target</entry></row><row><entry>Protein, HF-BE3</entry><entry>49300</entry><entry>42576</entry><entry>57690</entry><entry>HEK3 On Target</entry></row><row><entry>Plasmid, BE3</entry><entry>55008</entry><entry>55813</entry><entry>54310</entry><entry>HEK3 On Target</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>55199</entry><entry>11384</entry><entry>7659</entry><entry>HEK3 On Target</entry></row><row><entry>Control</entry><entry>63741</entry><entry>42878</entry><entry>48524</entry><entry>HEK3 On Target</entry></row><row><entry>Protein, BE3</entry><entry>104822</entry><entry>181792</entry><entry>161090</entry><entry>HEK3 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 1</entry></row><row><entry>Protein, HF-BE3</entry><entry>204580</entry><entry>175561</entry><entry>177303</entry><entry>HEK3 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 1</entry></row><row><entry>Plasmid, BE3</entry><entry>178584</entry><entry>152264</entry><entry>206863</entry><entry>HEK3 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 1</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>191297</entry><entry>138425</entry><entry>160789</entry><entry>HEK3 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 1</entry></row><row><entry>Control</entry><entry>190303</entry><entry>190061</entry><entry>190516</entry><entry>HEK3 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 1</entry></row><row><entry>Protein, BE3</entry><entry>146089</entry><entry>95113</entry><entry>135015</entry><entry>HEK3 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 2</entry></row><row><entry>Protein, HF-BE3</entry><entry>155947</entry><entry>136541</entry><entry>157991</entry><entry>HEK3 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 2</entry></row><row><entry>Plasmid, BE3</entry><entry>150036</entry><entry>128438</entry><entry>158905</entry><entry>HEK3 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 2</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>371077</entry><entry>123642</entry><entry>142562</entry><entry>HEK3 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 2</entry></row><row><entry>Control</entry><entry>130322</entry><entry>134545</entry><entry>141833</entry><entry>HEK3 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 2</entry></row><row><entry>Protein, BE3</entry><entry>145058</entry><entry>175338</entry><entry>161837</entry><entry>HEK3 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 3</entry></row><row><entry>Protein, HF-BE3</entry><entry>212337</entry><entry>178993</entry><entry>179887</entry><entry>HEK3 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 3</entry></row><row><entry>Plasmid, BE3</entry><entry>186452</entry><entry>166500</entry><entry>80441</entry><entry>HEK3 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 3</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>163732</entry><entry>118453</entry><entry>134719</entry><entry>HEK3 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 3</entry></row><row><entry>Control</entry><entry>131461</entry><entry>134470</entry><entry>155608</entry><entry>HEK3 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 3</entry></row><row><entry>Protein, BE3</entry><entry>41986</entry><entry>61678</entry><entry>67890</entry><entry>FANCF On Target</entry></row><row><entry>Protein, HF-BE3</entry><entry>41057</entry><entry>55850</entry><entry>86411</entry><entry>FANCF On Target</entry></row><row><entry>Plasmid, BE3</entry><entry>39114</entry><entry>48575</entry><entry>70074</entry><entry>FANCF On Target</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>41617</entry><entry>55638</entry><entry>75718</entry><entry>FANCF On Target</entry></row><row><entry>Control</entry><entry>68852</entry><entry>59422</entry><entry>81265</entry><entry>FANCF On Target</entry></row><row><entry>Protein, BE3</entry><entry>113462</entry><entry>80529</entry><entry>191344</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 1</entry></row><row><entry>Protein, HF-BE3</entry><entry>202662</entry><entry>233981</entry><entry>203024</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 1</entry></row><row><entry>Plasmid, BE3</entry><entry>208912</entry><entry>202044</entry><entry>107234</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 1</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>86494</entry><entry>113989</entry><entry>86807</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 1</entry></row><row><entry>Control</entry><entry>92255</entry><entry>72386</entry><entry>56661</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 1</entry></row><row><entry>Protein, BE3</entry><entry>96271</entry><entry>117442</entry><entry>84374</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 2</entry></row><row><entry>Protein, HF-BE3</entry><entry>105624</entry><entry>102312</entry><entry>105343</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 2</entry></row><row><entry>Plasmid, BE3</entry><entry>101002</entry><entry>98747</entry><entry>70052</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 2</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>308966</entry><entry>69787</entry><entry>83184</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 2</entry></row><row><entry>Control</entry><entry>99986</entry><entry>100344</entry><entry>100659</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 2</entry></row><row><entry>Protein, BE3</entry><entry>25524</entry><entry>182451</entry><entry>65388</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 3</entry></row><row><entry>Protein, HF-BE3</entry><entry>71858</entry><entry>75553</entry><entry>71785</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 3</entry></row><row><entry>Plasmid, BE3</entry><entry>60980</entry><entry>57360</entry><entry>78169</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 3</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>68316</entry><entry>34659</entry><entry>85718</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 3</entry></row><row><entry>Control</entry><entry>49685</entry><entry>57388</entry><entry>60418</entry><entry>FANCF Off Target</entry></row><row><entry /><entry /><entry /><entry /><entry>Site # 3</entry></row><row><entry>Protein, BE3</entry><entry>46981</entry><entry>90793</entry><entry>79439</entry><entry>EMX1 On Target</entry></row><row><entry>Protein, HF-BE3</entry><entry>58629</entry><entry>71186</entry><entry>61575</entry><entry>EMX1 On Target</entry></row><row><entry>Plasmid, BE3</entry><entry>70817</entry><entry>82736</entry><entry>75706</entry><entry>EMX1 On Target</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>77038</entry><entry>71123</entry><entry>78511</entry><entry>EMX1 On Target</entry></row><row><entry>Control</entry><entry>62183</entry><entry>48574</entry><entry>68439</entry><entry>EMX1 On Target</entry></row><row><entry>Protein, BE3</entry><entry>165905</entry><entry>257565</entry><entry>142888</entry><entry>EMX 1 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 1</entry></row><row><entry>Protein, HF-BE3</entry><entry>148339</entry><entry>151300</entry><entry>130712</entry><entry>EMX 1 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 1</entry></row><row><entry>Plasmid, BE3</entry><entry>101950</entry><entry>103226</entry><entry>203004</entry><entry>EMX 1 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 1</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>167969</entry><entry>175193</entry><entry>97010</entry><entry>EMX 1 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 1</entry></row><row><entry>Control</entry><entry>101476</entry><entry>150435</entry><entry>102327</entry><entry>EMX 1 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 1</entry></row><row><entry>Protein, BE3</entry><entry>136738</entry><entry>213438</entry><entry>118711</entry><entry>EMX 1 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 2</entry></row><row><entry>Protein, HF-BE3</entry><entry>123413</entry><entry>126114</entry><entry>109375</entry><entry>EMX 1 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 2</entry></row><row><entry>Plasmid, BE3</entry><entry>85576</entry><entry>86600</entry><entry>169592</entry><entry>EMX 1 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 2</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>140317</entry><entry>145738</entry><entry>137050</entry><entry>EMX 1 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 2</entry></row><row><entry>Control</entry><entry>84818</entry><entry>125139</entry><entry>85454</entry><entry>EMX 1 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 2</entry></row><row><entry>Protein, BE3</entry><entry>11940</entry><entry>36593</entry><entry>24946</entry><entry>EMX1 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 3</entry></row><row><entry>Protein, HF-BE3</entry><entry>26762</entry><entry>31566</entry><entry>36377</entry><entry>EMX1 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 3</entry></row><row><entry>Plasmid, BE3</entry><entry>32420</entry><entry>21547</entry><entry>14659</entry><entry>EMX1 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 3</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>31427</entry><entry>16592</entry><entry>17385</entry><entry>EMX1 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 3</entry></row><row><entry>Control</entry><entry>17385</entry><entry>28128</entry><entry>32717</entry><entry>EMX1 Off Target Site</entry></row><row><entry /><entry /><entry /><entry /><entry># 3</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><tbody valign="top"><row><entry>Murine Samples from NIH 3T3 cell treatment</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="5"><colspec colname="1" colwidth="63pt" align="left" /><colspec colname="2" colwidth="28pt" align="char" char="." /><colspec colname="3" colwidth="28pt" align="char" char="." /><colspec colname="4" colwidth="28pt" align="char" char="." /><colspec colname="5" colwidth="70pt" align="left" /><tbody valign="top"><row><entry>Plasmid, BE3</entry><entry>16641</entry><entry>102216</entry><entry>46361</entry><entry>On Target VEGFA</entry></row><row><entry /><entry /><entry /><entry /><entry>(Mus)</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>89330</entry><entry>126545</entry><entry>100993</entry><entry>On Target VEGFA</entry></row><row><entry /><entry /><entry /><entry /><entry>(Mus)</entry></row><row><entry>Protein, BE3</entry><entry>88998</entry><entry>81697</entry><entry>51124</entry><entry>On Target VEGFA</entry></row><row><entry /><entry /><entry /><entry /><entry>(Mus)</entry></row><row><entry>Protein, HF-BE3</entry><entry>128218</entry><entry>29193</entry><entry>131515</entry><entry>On Target VEGFA</entry></row><row><entry /><entry /><entry /><entry /><entry>(Mus)</entry></row><row><entry>Control</entry><entry>18767</entry><entry>38866</entry><entry>58985</entry><entry>On Target VEGFA</entry></row><row><entry /><entry /><entry /><entry /><entry>(Mus)</entry></row><row><entry>Plasmid, BE3</entry><entry>174782</entry><entry>167504</entry><entry>182565</entry><entry>CFD Off Target 1</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>167120</entry><entry>182520</entry><entry>192389</entry><entry>CFD Off Target 1</entry></row><row><entry>Protein, BE3</entry><entry>230569</entry><entry>212605</entry><entry>138144</entry><entry>CFD Off Target 1</entry></row><row><entry>Protein, HF-BE3</entry><entry>228668</entry><entry>211457</entry><entry>183370</entry><entry>CFD Off Target 1</entry></row><row><entry>Control</entry><entry>171738</entry><entry>191117</entry><entry>20879</entry><entry>CFD Off Target 1</entry></row><row><entry>Plasmid, BE3</entry><entry>206475</entry><entry>227332</entry><entry>206089</entry><entry>CFD Off Target 2</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>213809</entry><entry>203028</entry><entry>199078</entry><entry>CFD Off Target 2</entry></row><row><entry>Protein, BE3</entry><entry>215995</entry><entry>275754</entry><entry>249969</entry><entry>CFD Off Target 2</entry></row><row><entry>Protein, HF-BE3</entry><entry>250918</entry><entry>272063</entry><entry>241059</entry><entry>CFD Off Target 2</entry></row><row><entry>Control</entry><entry>193760</entry><entry>175959</entry><entry>246963</entry><entry>CFD Off Target 2</entry></row><row><entry>Plasmid, BE3</entry><entry>60388</entry><entry>126278</entry><entry>7328</entry><entry>CFD Off Target 3</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>89045</entry><entry>128508</entry><entry>5178</entry><entry>CFD Off Target 3</entry></row><row><entry>Protein, BE3</entry><entry>167195</entry><entry>330046</entry><entry>11163</entry><entry>CFD Off Target 3</entry></row><row><entry>Protein, HF-BE3</entry><entry>82120</entry><entry>309352</entry><entry>10393</entry><entry>CFD Off Target 3</entry></row><row><entry>Control</entry><entry>83204</entry><entry>176939</entry><entry>5661</entry><entry>CFD Off Target 3</entry></row><row><entry>Plasmid, BE3</entry><entry>192846</entry><entry>113709</entry><entry>171078</entry><entry>CFD Off Target 4</entry></row><row><entry>Plasmid, HF-BE3</entry><entry>205601</entry><entry>151434</entry><entry>188943</entry><entry>CFD Off Target 4</entry></row><row><entry>Protein, BE3</entry><entry>218194</entry><entry>181993</entry><entry>208398</entry><entry>CFD Off Target 4</entry></row><row><entry>Protein, HF-BE3</entry><entry>211966</entry><entry>148976</entry><entry>186838</entry><entry>CFD Off Target 4</entry></row><row><entry>Control</entry><entry>183933</entry><entry>130318</entry><entry>197476</entry><entry>CFD Off Target 4</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><tbody valign="top"><row><entry>Mouse Cochlea Samples</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="5"><colspec colname="1" colwidth="63pt" align="left" /><colspec colname="2" colwidth="28pt" align="char" char="." /><colspec colname="3" colwidth="28pt" align="char" char="." /><colspec colname="4" colwidth="28pt" align="char" char="." /><colspec colname="5" colwidth="70pt" align="left" /><tbody valign="top"><row><entry>Stria vascularies</entry><entry>37889</entry><entry>205706</entry><entry>62091</entry><entry>On Target (VEGFA)</entry></row><row><entry>Organ of Corti</entry><entry>148447</entry><entry>175004</entry><entry>29075</entry><entry>On Target (VEGFA)</entry></row><row><entry>Modiolus</entry><entry>182806</entry><entry>181382</entry><entry>61269</entry><entry>On Target (VEGFA)</entry></row><row><entry>Uninjected control</entry><entry>228222</entry><entry>241979</entry><entry>272759</entry><entry>On Target (VEGFA)</entry></row><row><entry>Stria vascularies</entry><entry>44457</entry><entry>244487</entry><entry>244646</entry><entry>CFD Off Target 1</entry></row><row><entry>Organ of Corti</entry><entry>136335</entry><entry>118318</entry><entry>34747</entry><entry>CFD Off Target 1</entry></row><row><entry>Modiolus</entry><entry>67176</entry><entry>209543</entry><entry>68699</entry><entry>CFD Off Target 1</entry></row><row><entry>Uninjected control</entry><entry>343100</entry><entry>342717</entry><entry>379015</entry><entry>CFD Off Target 1</entry></row><row><entry>Stria vascularies</entry><entry>72962</entry><entry>319883</entry><entry>265793</entry><entry>CFD Off Target 2</entry></row><row><entry>Organ of Corti</entry><entry>198456</entry><entry>131430</entry><entry>60530</entry><entry>CFD Off Target 2</entry></row><row><entry>Modiolus</entry><entry>92014</entry><entry>251509</entry><entry>81413</entry><entry>CFD Off Target 2</entry></row><row><entry>Uninjected control</entry><entry>399138</entry><entry>345965</entry><entry>483920</entry><entry>CFD Off Target 2</entry></row><row><entry>Stria vascularies</entry><entry>8325</entry><entry>80322</entry><entry>142556</entry><entry>CFD Off Target 3</entry></row><row><entry>Organ of Corti</entry><entry>81014</entry><entry>45976</entry><entry>1810</entry><entry>CFD Off Target 3</entry></row><row><entry>Modiolus</entry><entry>9928</entry><entry>75555</entry><entry>11341</entry><entry>CFD Off Target 3</entry></row><row><entry>Uninjected control</entry><entry>399138</entry><entry>345965</entry><entry>483920</entry><entry>CFD Off Target 3</entry></row><row><entry>Stria vascularies</entry><entry>232194</entry><entry>397770</entry><entry>554054</entry><entry>CFD Off Target 4</entry></row><row><entry>Organ of Corti</entry><entry>313472</entry><entry>285302</entry><entry>176872</entry><entry>CFD Off Target 4</entry></row><row><entry>Modiolus</entry><entry>230105</entry><entry>371399</entry><entry>258142</entry><entry>CFD Off Target 4</entry></row><row><entry>Uninjected control</entry><entry>524503</entry><entry>637946</entry><entry>624709</entry><entry>CFD Off Target 4</entry></row><row><entry namest="1" nameend="5" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><tbody valign="top"><row><entry>Zebrafish samples</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="5"><colspec colname="1" colwidth="63pt" align="left" /><colspec colname="2" colwidth="28pt" align="center" /><colspec colname="3" colwidth="28pt" align="center" /><colspec colname="4" colwidth="28pt" align="center" /><colspec colname="5" colwidth="70pt" align="left" /><tbody valign="top"><row><entry /><entry>1</entry><entry>2</entry><entry>3</entry><entry>Amplicon</entry></row><row><entry namest="1" nameend="5" align="center" rowsep="1" /></row><row><entry>Treated zebrafish</entry><entry>72355</entry><entry>49498</entry><entry>81061</entry><entry>TYR 1</entry></row><row><entry>Scrambled sgRNA</entry><entry>107919</entry><entry>98502</entry><entry>92429</entry><entry>TYR 1</entry></row><row><entry>Treated zebrafish</entry><entry>51434</entry><entry>48014</entry><entry>41547</entry><entry>TYR 2</entry></row><row><entry>Scrambled sgRNA</entry><entry>61466</entry><entry>62374</entry><entry>66765</entry><entry>TYR 2</entry></row><row><entry>Treated zebrafish</entry><entry>6487</entry><entry>57247</entry><entry>75883</entry><entry>TYR 3</entry></row><row><entry>Scrambled sgRNA</entry><entry>64596</entry><entry>71234</entry><entry>75624</entry><entry>TYR 3</entry></row><row><entry namest="1" nameend="5" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><tbody valign="top"><row><entry>Mouse Cochlea Samples - treated with unrealated sgRNA</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="147pt" align="center" /><colspec colname="2" colwidth="70pt" align="center" /><tbody valign="top"><row><entry>Sample</entry><entry /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="1" colwidth="56pt" align="center" /><colspec colname="2" colwidth="28pt" align="center" /><colspec colname="3" colwidth="63pt" align="center" /><colspec colname="4" colwidth="70pt" align="left" /><tbody valign="top"><row><entry>Stria</entry><entry>Cortii</entry><entry>Modiolus</entry><entry>Amplicon</entry></row><row><entry namest="1" nameend="4" align="center" rowsep="1" /></row><row><entry>537459</entry><entry>249767</entry><entry>389274</entry><entry>On Target (VEGFA)</entry></row><row><entry namest="1" nameend="4" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0914Finally, these developments were applied to achieve DNA-free, high-specificity base editing in mice. To maximize the likelihood of observing on- and off-target base editing in vivo, the highly repetitive sgRNA targeting VEGFA site 2 was used; conveniently, the murine and human genomes are identical at this target site.

0915Using cultured murine NIH/3T3 cells, it was confirmed that BE3 protein delivery yielded efficient on-target base editing at this locus 34±11% (<figref idref="DRAWINGS">FIG. 133A</figref>; all editing percentages in this paragraph represent mean±s.d. for n=3 biological replicates). The Cutting Frequency Determinant (CFD) algorithm^29,34was used to predict off-target loci in the mouse genome associated with the VEGFA site 2 sgRNA (Table 18). Using cultured NIH/3T3 cells, it was confirmed that two of the top four predicted off-target loci are indeed modified by plasmid delivery of BE3 in cultured murine cells (CFD off-target locus 1, 9±5% editing; and CFD off-target locus 4, 3±2% editing; <figref idref="DRAWINGS">FIG. 133B-133E</figref>). Consistent with the results from human cells, protein delivery of BE3 reduced off-target editing to levels similar to that of negative controls (<figref idref="DRAWINGS">FIGS. 133C and 133E</figref>). The mean base editing specificity ratio for CFD off-target loci 1 and 4 increased from 28±13 for plasmid delivery of BE3 to ≥780±300 for protein delivery of BE3 (values represent mean±s.e.m.; n=3 biological replicates).

0916<tables id="TABLE-US-00176" num="00176"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="329pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 18</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Protospacer and PAM sequences for the predicted off-target loci in the mouse</entry></row><row><entry>genome associated with the VEGFA site 2 sgRNA. CFD scores ⁴⁶ were calculated</entry></row><row><entry>using CRISPOR ⁴⁷.</entry></row><row><entry>Positions in the off-target protospacers that differ from the on-target</entry></row><row><entry>sequence are underlined.</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="5"><colspec colname="1" colwidth="56pt" align="left" /><colspec colname="2" colwidth="105pt" align="left" /><colspec colname="3" colwidth="28pt" align="center" /><colspec colname="4" colwidth="35pt" align="center" /><colspec colname="5" colwidth="105pt" align="left" /><tbody valign="top"><row><entry /><entry /><entry>SEQ</entry><entry>CFD</entry><entry /></row><row><entry>Site</entry><entry>Sequence</entry><entry>ID NO</entry><entry>score</entry><entry>Description of locus</entry></row><row><entry namest="1" nameend="5" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="5"><colspec colname="1" colwidth="56pt" align="left" /><colspec colname="2" colwidth="105pt" align="left" /><colspec colname="3" colwidth="28pt" align="center" /><colspec colname="4" colwidth="35pt" align="char" char="." /><colspec colname="5" colwidth="105pt" align="left" /><tbody valign="top"><row><entry>On-target</entry><entry>GACCCCCTCCACCCCGCCTCCGG</entry><entry>497</entry><entry /><entry>VEGFA site 2</entry></row><row><entry></entry></row><row><entry>Off-target 1</entry><entry><u style="single">TC</u>CCCCCTCCACCCC<u style="single">A</u>CCTCCGG</entry><entry>498</entry><entry>0.7857</entry><entry>intergenic:mmu-mir-21c-</entry></row><row><entry /><entry /><entry /><entry /><entry>Nrp1/Mir1903</entry></row><row><entry>Off-target 2</entry><entry><u style="single">TG</u>CCCAC<u style="single">CT</u>CACCCC<u style="single">G</u>CCTCTGG</entry><entry>499</entry><entry>0.65</entry><entry>intron:Vipr1</entry></row><row><entry></entry></row><row><entry>Off-target 3</entry><entry>G<u style="single">C</u>CCC<u style="single">T</u>C<u style="single">C</u>CA<u style="single">A</u>CCCC<u style="single">A</u>CCTCTGG</entry><entry>500</entry><entry>0.6323</entry><entry>intron:Nos1ap</entry></row><row><entry></entry></row><row><entry>Off-target 4</entry><entry><u style="single">C</u>ACCCCC<u style="single">CT</u>CACCCCGCCTCAGG</entry><entry>501</entry><entry>0.625</entry><entry>intergenic:Unc5b-mmu-</entry></row><row><entry /><entry /><entry /><entry /><entry>mir-6408</entry></row><row><entry namest="1" nameend="5" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0917To establish DNA-free base editing in mice, BE3:sgRNA complexes were combined with LIPOFECTAMINE® 2000 transfection reagent (<figref idref="DRAWINGS">FIG. 132B</figref>) and intracochlear injections were performed into mouse pups at P1-P2. Injected cochlear tissues were harvested 3-4 days post-injection and micro-dissected into 5-7 samples per cochlear region. Control cochlea from uninjected mice were harvested simultaneously. Genomic DNA was extracted from the harvested tissue, amplified by qPCR to late-exponential phase, and subjected to high-throughput DNA sequencing to measure C→T conversion. Although it is impossible to quantitate base editing efficiency among treated cells because it is not possible to retrieve DNA exclusively from cells exposed to base editor protein, unambiguous base editing was observed from tissue in three regions of the cochlea: the basal end of the organ of <i>Corti</i>, the stria vascularis and the modiolus (<figref idref="DRAWINGS">FIGS. 132C-132D</figref>). No significant indel formation was detected in treated tissue samples (&lt;0.1% indels; <figref idref="DRAWINGS">FIG. 134B</figref>).

0918The percentage of cochlear cells containing target C→T conversion (<figref idref="DRAWINGS">FIG. 132C</figref>) was significantly lower than that observed in treated NIH/3T3 cells in culture (<figref idref="DRAWINGS">FIG. 133A</figref>), consistent with the highly localized nature of lipid-based protein delivery and the inability to isolate DNA exclusively from cells exposed to base editor. Nonetheless, local delivery offers key advantages for accessible applications, including control over which cell types are edited, and ease of preparation and administration.

0919Finally, off-target editing following intracochlear injection of BE3:sgRNA:lipid complexes was analyzed. Analysis of all four predicted off-target loci, including the confirmed off-target sites CFD locus 1 and CFD locus 4, in genomic DNA from the cochlear tissue of mice injected with the BE3:VEGFA site 2 sgRNA:lipid complex revealed no detectable C→T conversion or indel formation above that observed in untreated controls samples for any of the off-target loci tested (<figref idref="DRAWINGS">FIG. 134A</figref>).

0920Together, these in vivo base editing results establish a virus-free, DNA-free strategy for the precise conversion of individual nucleotides in the genomic DNA of animals with high DNA sequence specificity.

DISCUSSION

0921The strategies developed and implemented in this study expand the utility and applicability of base editing by removing or reducing off-target base editing and establishing a DNA-free delivery method that supports in vivo base editing. Protein delivery improves base editing specificity in human and murine cells compared with plasmid delivery of the same constructs (<figref idref="DRAWINGS">FIGS. 137, 130, and 133</figref>), and enables specific base editing in zebrafish and in the mouse cochlea (<figref idref="DRAWINGS">FIG. 132</figref>).

0922A high-fidelity base editor was generated by installing into BE3 mutations known to enhance the DNA specificity of Cas9²⁰. The installation of these mutations into Cas9 was reported to result in undetectable indel formation at off target loci associated with non-repetitive sgRNAs, including the EMX1 locus interrogated here (<figref idref="DRAWINGS">FIG. 127A</figref>)²⁰. The specificity enhancements observed in HF-BE3, while substantial, were more modest; HF-BE3 exhibited detectable off-target base editing at both repetitive and non-repetitive loci when delivered as plasmid DNA into mammalian cells (<figref idref="DRAWINGS">FIGS. 127A, 127D, 133C, and 133E</figref>). It is tempting to speculate that this specificity enhancement difference may arise from the fact that base editing, unlike Cas9-mediated indel formation, does not require DNA cleavage but only necessitates DNA-binding and R-loop formation¹⁴, and some of the enhanced specificity of HF-Cas9 may arise from impaired DNA cleavage at already-bound off-target loci.

0923In a second attempt to reduce off-target base editing, it was demonstrated that RNP delivery of base editors leads to decoupling of on- and off-target editing (<figref idref="DRAWINGS">FIG. 128B-128C</figref>). RNP delivery ablated off-target editing at non-repetitive sites while maintaining on-target editing comparable to plasmid delivery (<figref idref="DRAWINGS">FIGS. 130A-130C and 128A</figref>), and greatly reduced off-target editing even at the highly repetitive VEGFA site 2 (<figref idref="DRAWINGS">FIG. 130D</figref>). RNP delivery of base editors may be especially useful for in vivo editing applications in which cellular dosage is typically difficult to control or characterize.

0924RNP delivery of Cas9 coupled with delivery of a donor DNA template has previously been used to perform HDR-based genome editing in mammalian cells. These approaches, however, remain limited by low efficiency, cell-state dependence, and indel formation efficiencies typically exceeding those of desired HDR outcomes, especially for point mutation correction^29,30,32,35DNA-free base editing, in contrast, generates a substantial excess of edited product relative to stochastic indels both in vivo and in cells (<figref idref="DRAWINGS">FIGS. 132A, 134A, and 134B</figref>). To the best of the inventors' knowledge, RNP delivery of base editors represents the first strategy for generating specific and precise modifications to genomic DNA without requiring exogenous DNA.

0000Methods

0000Cloning of Plasmids

0925The plasmids in this study were generated by USER cloning. Phusion U Hot Start polymerase (Thermo Fisher) was used to install point mutations and construct protein expression plasmids from previously reported constructs³⁶. Protein sequences are listed in the Supplementary Information, and plasmids for expression of BE3 and HF-BE3 are available from Addgene.

0000Expression and Purification of BE3 and HF-BE3

0926BL21 Star (DE3)-competent <i>E. coli </i>cells were transformed with plasmids encoding the bacterial codon optimized base editors with a His6 N-terminal purification tag. A single colony was grown overnight in Luria-Bertani (LB) broth containing 50 μg mL⁻¹kanamycin at 37° C. The cells were diluted 1:200 into 2 L of the same media and grown at 37° C. until OD₆₀₀=0.70-0.75. The cultures were incubated on ice for 60 min and protein expression was induced with 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG, GoldBio). Expression was sustained for 14-16 h with shaking at 18° C. The subsequent purification steps were carried out at 4° C. Cells were collected by centrifugation at 6,000 g for 20 min and resuspended in cell collection buffer (100 mM tris(hydroxymethyl)-aminomethane (Tris)-HCl, pH 8.0, 1 M NaCl, 20% glycerol, 5 mM tris(2-carboxyethyl)phosphine (TCEP; GoldBio), 0.4 mM phenylmethane sulfonyl fluoride (PMSF; Sigma Aldrich) and 1 cOmplete, EDTA-free protease inhibitor pellet (Roche) per 50 mL buffer used). Cells were lysed by sonication (6 min total, 3 s on, 3 s off) and the lysate cleared by centrifugation at 25,000 g (20 min).

0927The cleared lysate was incubated with His-Pur nickel nitriloacetic acid (nickel-NTA) resin (1 mL resin per litre of culture, Thermo Fisher) with rotation at 4° C. for 60-90 min. The resin was washed with 20 column volumes of cell collection buffer before bound protein was eluted with elution buffer ((100 mM tris(hydroxymethyl)-aminomethane (Tris)-HCl, pH 8.0, 0.5 M NaCl, 20% glycerol, 5 mM tris (2-carboxyethyl) phosphine (TCEP; GoldBio), 200 mM imidazole). The resulting protein fraction was further purified on a 5 mL Hi-Trap HP SP (GE Healthcare) cation exchange column using an Akta Pure FPLC. Protein-containing fractions were concentrated using a column with a 100,000 kDa cutoff (Millipore) centrifuged at 3,000 g and the concentrated solution was sterile filtered through an 0.22 μm PVDF membrane (Millipore).

0928After sterile filtration, proteins were quantified with Reducing Agent Compatible Bicinchoninic acid (BCA) assay (Pierce Biotechnology), snap-frozen in liquid nitrogen and stored in aliquots at −80° C. Sequences of expressed proteins are listed in Supplementary Note 2.

0000In Vitro Transcription of sgRNA

0929Linear DNA fragments containing the T7 RNA polymerase promoter sequence upstream of the desired 20 bp sgRNA protospacer and the sgRNA backbone were generated by PCR (Q5 Hot Start MasterMix, New England Biolabs) using primers as listed in the Supplementary Information and concentrated on minelute columns (Qiagen). sgRNA was transcribed with the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs) at 16° C. for 14-16 h with 1 μg of linear template per 20 μL reaction. sgRNA was purified using the MEGAClear Transcription Clean Up Kit (Thermo Fisher), according to the manufacturer's instructions. Purified sgRNAs were stored in aliquots at −80° C.

0000In Vitro Deamination Assays

0930Sequences of DNA oligonucleotides used as templates for the in vitro deamination assay are shown in Supplementary Note 3. All oligonucleotides were purchased from IDT. Single-stranded oligonucleotides synthesized with complementary sequences were combined (5 μL of a 100 μM solution) in Tris buffer pH 8.0 and annealed by heating to 95° C. for 5 min, followed by a gradual cooling to 37° C. at a rate of 0.1° C. second⁻¹to generate 79 base pair (bp) dsDNA substrates. Freshly thawed base-editor proteins (2 μM final concentration in a 10 μL reaction volume) were complexed with the indicated sgRNA (2.2 μM final concentration) in Reaction Buffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA, 10 mM MgCl₂)³⁷for five minutes at room temperature. Annealed dsDNA substrates were then added to a final concentration of 250 nM. The reaction proceeded for 30 min at 37° C. before protein denaturation was performed by heating for 5 min at 99° C. Addition of PB buffer (Qiagen, 100 μL) and isopropanol (25 μL) ensured protein was dissociated from the substrate DNA. DNA was purified with Minelute columns (Qiagen) and the resulting products amplified to the top of the linear range with 15 cycles of qPCR (12 ng input DNA, 50 μL reaction volume) using a U-tolerant polymerase (Phusion U Hot Start, ThermoFisher) and primers as listed in the Supplementary Information. Amplified DNA was purified using RapidTip2 (Diffinity Genomics) and barcoded with a second round of PCR (8 cycles, 5 ng input) before being prepared for sequencing on an Illumina MiSeq as described below.

0000Purification and Sequencing of Genomic DNA

0931Genomic DNA was isolated using Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter) according to the manufacturer's instructions. For the first PCR, DNA was amplified to the top of the linear range using Q5 Hot Start DNA Polymerase (NEB), according to the manufacturer's instructions but with the addition of 3% DMSO and SYBR Gold Nucleic Acid Stain (Thermo Fisher). For all amplicons, the PCR protocol used was an initial heating step of 2 min at 98° C. followed by an optimized number of amplification cycles (12 s at 98° C., 25 s at 61° C., 30 s at 72° C.). For zebrafish and for transfected cell samples, 30 ng of input DNA was used in a 50 μL reaction, for cochlear samples 20 ng was used in a 25 μL reaction. qPCR was performed to determine the optimal cycle number for each amplicon. Amplified DNA was purified using RapidTip2 (Diffinity Genomics) and barcoded with a further PCR (8 cycles, 5 ng input). The unique forward and reverse primers used in the first-round PCR contained a constant region 5′ to the annealing region, (forward: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNN-3′ (SEQ ID NO: 502), reverse: 5′-TGGAGTTCAGACGTGTGCTCTTCCGATCT-3′(SEQ ID NO: 503)) which facilitated binding of barcoding primers to amplified DNA for a second-round PCR.

0932The second-round PCR used primers with three regions: a 5′ constant region allowing the amplicon to bind to the Illumina flow cell (italicized), an 8-base barcoding region (X), and a 3′ constant region allowing the barcoding primer to bind to the first-round PCR amplicon (in bold). Examples of primer sequences are:

0933<tables id="TABLE-US-00177" num="00177"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 504)</entry></row><row><entry>forward: 5′-<i>AATGATACGGCGACCACCGAGATCTACAC</i>XXXXXXXX<b>A</b></entry></row><row><entry></entry></row><row><entry><b>CACTCTTTCCCTACACGAC-3′</b></entry></row><row><entry></entry></row><row><entry>(SEQ ID NO: 505)</entry></row><row><entry>reverse: 5′-CAAGCAGAAGACGGCATACGAGATXXXXXXX<b>GTGACTG</b></entry></row><row><entry></entry></row><row><entry><b>GAGTTCAGACGTGTGCTCTTC-3′</b></entry></row></tbody></tgroup></table></tables>

0934Sequencing adapters and dual-barcoding sequences are based on the TruSeq Indexing Adapters (Illumina). Barcoded samples were pooled and purified by gel extraction (Qiagen), and then purified using Ampure beads (Beckman Coulter) before quantification using the Qubit dsDNA HS Kit (Thermo Fisher) and qPCR (KAPA BioSystems) according to the manufacturer's instructions. Sequencing of pooled samples was performed using a single-end read from 180-250 bases (depending on the amplicon size) on the MiSeq (Illumina) according to the manufacturer's instructions.

0935Sequences of oligonucleotides used for PCR amplification are shown in Supplementary Note 3. All oligonucleotides were obtained from IDT. The optimized number of PCR cycles for each amplicon in this study are as follows: VEGFA site 2 human genomic DNA (annealing temperature was 61° C. for 25 seconds for all extension steps): on-target: 29 cycles, off-target #1: 32 cycles, off-target #2: 28 cycles, off-target #3: 27 cycles, off-target #4: 27 cycles, VEGFA site 2 murine genomic DNA: on-target: 31 cycles, off-targets #1, #2, #3 and #4: 31 cycles. HEK293 site 3: off-targets #1: 29 cycles, off-target #2: 28 cycles, off-target #3: 28 cycles. FANCF off-target #1: 29 cycles, off-target #2: 28 cycles, off-target 3: 28 cycles. EMX1 off-targets #1, #2 and #3: 28 cycles. TYR1, TYR2 and TYR3 sgRNAs for amplification of zebrafish DNA: 32 cycles. Optimized protocols for the on-target amplification of the EMX1, FANCF, and HEK293 site 3 loci were followed as previously described¹⁴.

0000Analysis and Alignment of Genomic DNA Sequencing Reads

0936Sequencing reads were analyzed as previously described¹⁴. In brief, sequencing reads were demultiplexed using MiSeq Reporter (Illumina), and individual FASTQ files were analyzed with a previously reported custom Matlab script¹⁴. Reads were aligned to the reference sequence using the Smith-Waterman algorithm. Base calls with Q-scores below 30 were replaced with a placeholder nucleotide (N). This quality threshold results in nucleotide frequencies with an expected error rate of 1 in 1,000. Indel frequencies were quantified with a previously published custom Matlab script which counts indels which occurring in a 30-base window around the nCas9 cleavage site and are a minimum of 2-base insertions or deletions¹⁴. Indels were defined as detectable if there was a significant difference (Student's two-tailed t-test, p&lt;0.05) between indel formation in the treated sample and untreated control.

0937For one of the sequenced amplicons, CFD off-target #3, associated with VEGFA site 2 sgRNA in the murine genome, it was not possible to accurately measure indel formation. The protospacer at this locus is directly preceded by 12 guanine bases, which makes PCR and high-throughput sequencing of this site prone to random insertion or deletions; deletion rates as high as 20% of sequencing reads were observed in multiple independent untreated control samples. Since no significant base editing was detected at this off-target locus under any treatment conditions (<figref idref="DRAWINGS">FIGS. 132 and 133</figref>), it is suspected that indel formation is also negligible at this locus.

0938A phred.II Q30 score corresponds to an estimated 99.9% accuracy in basecalling³⁸. A 0.1% probability of incorrect base calling at a given position corresponds to a lower limit for base calling of 0.1/4=0.025% if it is assumed base call errors are randomly distributed across the four bases. C→T editing percentages that fell beneath this threshold were classified as undetectable. Spontaneous deamination³⁹or polymerase error during PCR can also introduce artefactual C→T edits. In order to distinguish base editor-induced C→T editing from artefactual C+T editing rates, untreated control cells were sequenced for each amplicon and it was calculated whether the C→T editing under a particular condition was statistically significant using the Student's two-tailed t-test with p<0.05 as the threshold. Off-target sites with statistically significant editing rates >0.025% were considered measureable. The number of aligned and quality filtered reads for each sample has been included in Table 17.

0000Statistical Analyses of Genomic DNA Sequence Alignments

0939Unless otherwise noted, mean values cited throughout the main text are representative of n≥3 independent biological replicates and the mean±standard deviation has been stated.

0940The statistical analysis of the high-throughput sequencing data displayed in <figref idref="DRAWINGS">FIGS. 2 and 3</figref> was performed by comparing on- and off-target editing percentages in treated samples to any editing measured in a negative control sample (untreated). The Student's two-tailed t test was used, and individual p-values are shown in Table 16. * p≤0.05, ** p≤0.01 and *** p≤0.001. When editing was below the detection limit (0.025%), significance was not calculated; all untreated control samples showed undetectable editing.

0941For <figref idref="DRAWINGS">FIG. 128A</figref>, mean on-target base editing was calculated by averaging editing of cytosines in the base editing activity window (C₄-C₈for HEK293 site 3 and EMX1, C₄-C₉for FANCF and VEGFA site 2).

0942To account for sgRNA-dependent differences in base editing activity, the a base editing:indel ratio was calculated (<figref idref="DRAWINGS">FIG. 130B</figref>). This ratio was generated by dividing the percentage of HTS reads with a C→T conversion (averaged across the base editing window for each site) by the percentage of HTS reads containing an indel. As described above, if the off-target editing for a particular locus was below the limit of detection it was conservatively assumed the estimated upper bound of the detection method (0.025%) for the purpose of calculating specificity ratios.

0000Data Analysis of In Vitro Edited DNA

0943Sequencing reads were automatically demultiplexed using MiSeq Reporter (Illumina.). Quality filtering was performed using the online package usegalaxy.org⁴⁰. Individual bases with an Illumina quality score less than or equal to 30 were converted to the placeholder nucleotide ‘N’ using FASTQ Groomer followed by FASTA Masker⁴¹. The resulting quality-filtered FASTQ files were subsequently analysed with a custom python script provided in Supplementary Note 1. Sequencing reads were scanned for exact matches to two 14-base sequences that flank both sides of the target DNA sequence. If no exact matches were found, the read was excluded from analysis. If both 14-base sequences were located and the length of the sequence between them was equal to the expected protospacer length (20 bases), the protospacer sequence found between the flanking regions was saved and the bases called by high-throughput sequencing at each site within the protospacer were tallied.

0000Cell Culture

0944Both HEK293T (ATCC CRL-3216) and NIH/3T3 (ATCC CRL-1658) were maintained in Dulbecco's Modified Eagle's Medium plus GlutaMax (ThermoFisher) supplemented with 10% (v/v) fetal bovine serum (FBS), at 37° C. with 5% CO₂. Cells were obtained from ATCC and were authenticated and verified to be free of <i>mycoplasma </i>by ATCC upon purchase.

0000Plasmid Transfection of Base Editors into HEK293T Cells

0945HEK293T cells were seeded on 48-well collagen-coated BioCoat plates (Corning) in antibiotic free medium and transfected at approximately 70% confluency. Unless otherwise noted, 750 ng of BE and 250 ng of sgRNA expression plasmids were transfected using 1.5 μl of LIPOFECTAMINE® 2000 transfection reagent (Thermo Fisher) per well according to the manufacturer's protocol.

0000Protein Transfection of Base Editors into HEK293T Cells

0946HEK293T cells were seeded on 48-well collagen-coated BioCoat plates (Corning) in 250 μL antibiotic free medium and transfected at approximately 70% confluency. Base editor protein and was incubated with 1.1× molar excess of the necessary sgRNA at room temperature for 5 min. The complex was then incubated with 1.5 μL LIPOFECTAMINE® 2000 transfection reagent (Thermo Fisher) and transfected according to the manufacturer's protocol for plasmid delivery. Unless otherwise noted, BE protein was added to a final concentration of 200 nM (based on a total well volume of 275 μL).

0000Plasmid Transfection of Base Editors into NIH/3T3 Cells

0947NIH/3T3 cells were seeded on 48-well collagen-coated BioCoat plates (Corning) in antibiotic-free DMEM medium and transfected at approximately 75% confluency. Unless otherwise noted, 600 ng of BE and 200 ng of sgRNA expression plasmids were transfected using 1.4 μL of LIPOFECTAMINE® 3000 transfection reagent with 1 μL of P3000 reagent (Thermo Fisher) per well according to the manufacturer's protocol.

0000Protein Transfection of Base Editors into NIH/3T3 Cells

0948NIH/3T3 cells were seeded on 48-well collagen-coated BioCoat plates (Corning) in antibiotic free DMEM medium and transfected at approximately 75% confluency. Base editor proteins were incubated with 1.1-fold molar excess of the indicated sgRNA at 25° C. for 5 min. The complex was then incubated with 1.4 μL LIPOFECTAMINE® 3000 transfection reagent (Thermo Fisher) and transfected according to the manufacturer's protocol for plasmid delivery. P3000 reagent was not used because its addition lead to protein precipitation and a reduction in base editing efficiency. Unless otherwise noted, BE protein was added to a final concentration of 400 nM (based on a total well volume of 275 μL).

0000Intracochlear Delivery of BE3 Protein:Guide RNA Encapsulated in Cationic Lipid

0949All animal experiments were approved by the Institutional Animal Care and the Use Committee of the Massachusetts Eye and Ear Infirmary. Intracochlear delivery was performed in P1-P2 mice of a mixed genetic background as described previously⁴². Mice were anesthetized by lowering body temperature before the surgical procedure. A postauricular incision was made near the right ear, and the bulla was lifted to expose the cochlea. BE3 protein (57.7 μM) was pre-complexed with the sgRNA (100 μM) in a 1:1.1 molar ratio and then mixed with LIPOFECTAMINE® 2000 transfection reagent (Thermo Fisher) in a 1:1 volumetric ratio. The resulting solution (1.2-1.5 μL) was injected with a glass pipette (end diameter, 5 μm) through the cochlear capsule into scala media at the cochlear basal turn that attached to a nanoliter micropump (WPI, UMP3+Micro4+NanoFil) at the rate of 250 nL min⁻¹. After injection, the incision was closed and the mice were brought onto a heating pad to recover. After 3-4 days, the cochlea of mouse was dissected into the organ of <i>Corti</i>, stria vascularis, and modiolus. Each tissue was further micro-dissected into between 5 and 7 separate pieces and DNA extraction was performed separately for each sample, followed by high-throughput sequencing as described above. The data presented in <figref idref="DRAWINGS">FIG. 132</figref> and <figref idref="DRAWINGS">FIG. 134</figref> show sequencing data resulting from extraction of one micro-dissected sample for each cochlear region.

0000Microinjection of BE3 Protein:Guide RNA into Zebrafish Embryo

0950Zebrafish (Tuebingen strain) were maintained under standard conditions in compliance with internal regulatory review at Boston Children's Hospital. One-cell stage zebrafish embryos were injected with approximately 2 nL of BE3 protein pre-complexed with the appropriate sgRNA or an unrelated sgRNA control in a 1:1 molar ratio (4.5 μM final concentration). Four days post-fertilization, DNA was extracted from larvae as previously described⁴³in 50 mM NaOH for 30 minutes at 95° C. and the resulting solution was neutralized with Tris-HCl. Genomic DNA was quantified, amplified by PCR, and sequenced as described above.

0000Protein Gel Analyses

0951All protein gels shown were precast 4-12% polyacrylamide Bis-Tris Plus (Thermo Fisher). They were run in MOPS buffer (Thermo Fisher) at 180 V for 50 min. Samples were prepared for loading by heating to 99° C. in 100 mM DTT and 1× lithium dodecyl sulfate (LDS) Sample Buffer for denaturation (Thermo Fisher) for 10 min. Gels were stained using Instant Blue Protein Stain (Expedion) according to manufacturer's instructions.

0952For cell lysate analysis, 2 mL of post-induction overnight culture was pelleted at 15,000 g before lysis in 100 μL B-PER (Thermo Fisher) according to the manufacturer's instructions.

0000Data Availability

0953High-throughput sequencing data that support the findings of this study have been deposited in the NCBI Sequence Read Archive database under Accession Number SRP097884. Plasmids encoding HF-BE3 and BE3 for protein expression, as well as HF-BE3 for mammalian expression, are available from Addgene with Accession IDs 87439 (pCMV-HF-BE3), 87438 (pET42b-HF-BE3), 87437 (pET42b-BE3).

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Supplementary Information

1001<tables id="TABLE-US-00178" num="00178"><table frame="none" colsep="0" rowsep="0" pgwide="1"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="266pt" align="center" /><thead><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Supplementary Note 1: Python script used to analyze quality-filtered in vitro-edited DNA.</entry></row><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="14pt" align="left" /><colspec colname="2" colwidth="252pt" align="left" /><tbody valign="top"><row><entry> 1.</entry><entry>from_future_import print_function</entry></row><row><entry> 2.</entry><entry>from_future_import division</entry></row><row><entry> 3.</entry><entry /></row><row><entry> 4.</entry><entry>import Bio #This will import the BioPython suite</entry></row><row><entry> 5.</entry><entry>from Bio import SeqIO #Necessary to read/write sequence handles</entry></row><row><entry> 6.</entry><entry>from Bio.Seq import Seq</entry></row><row><entry> 7.</entry><entry>import os</entry></row><row><entry> 8.</entry><entry>import collections</entry></row><row><entry> 9.</entry><entry>import csv</entry></row><row><entry>10.</entry><entry /></row><row><entry>11.</entry><entry>inputfile = “please_specify_your_input_file_here_containing_filtered_reads” #specify </entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="266pt" align="left" /><tbody valign="top"><row><entry>the filenames that contain sequences</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="14pt" align="left" /><colspec colname="2" colwidth="252pt" align="left" /><tbody valign="top"><row><entry>12.</entry><entry>filenames = [ ]</entry></row><row><entry>13.</entry><entry /></row><row><entry>14.</entry><entry>for file in os.listdir(inputfile):</entry></row><row><entry>15.</entry><entry> if file.endswith(“.fastqsanger”):</entry></row><row><entry>16.</entry><entry>  filenames.append(file)</entry></row><row><entry>17.</entry><entry /></row><row><entry>18.</entry><entry>spacer = [ ]</entry></row><row><entry>19.</entry><entry>list_of_filenames = [ ]</entry></row><row><entry>20.</entry><entry /></row><row><entry>21.</entry><entry>for file in filenames:</entry></row><row><entry>22.</entry><entry> site = { }</entry></row><row><entry>23.</entry><entry> output = open(file + “.txt”, “w”)</entry></row><row><entry>24.</entry><entry> list_of_filenames.append(file + “.txt”) #allows calling of the txt files that come</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="266pt" align="left" /><tbody valign="top"><row><entry>from fastq files later</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="14pt" align="left" /><colspec colname="2" colwidth="252pt" align="left" /><tbody valign="top"><row><entry>25.</entry><entry> for rec in SeqIO.parse(file, “fastq”):</entry></row><row><entry>26.</entry><entry>  split1=rec.seq.tostring( ).split(“GTTCGCGGCGATCG”) #14-</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="266pt" align="left" /><tbody valign="top"><row><entry>base pair constant_region_before_protospacer</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="14pt" align="left" /><colspec colname="2" colwidth="252pt" align="left" /><tbody valign="top"><row><entry>27.</entry><entry>  if len(split1)>=2:</entry></row><row><entry>28.</entry><entry>   split2=split1[1].split(“TGGATCGCCTGGCA”) #14-</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="266pt" align="left" /><tbody valign="top"><row><entry>base pairc constant_region_after_protospacer</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="14pt" align="left" /><colspec colname="2" colwidth="252pt" align="left" /><tbody valign="top"><row><entry>29.</entry><entry>   site=split2[0]</entry></row><row><entry>30.</entry><entry>   if len(site)==20:</entry></row><row><entry>31.</entry><entry>    output.write(site + “\n”)</entry></row><row><entry>32.</entry><entry /></row><row><entry>33.</entry><entry>BASES = ‘ATGCN’</entry></row><row><entry>34.</entry><entry>UNRECOGNIZED = ‘X’</entry></row><row><entry>35.</entry><entry>BASE_SEPERATOR = dict(zip(BASES, ‘,,,,\n’))</entry></row><row><entry>36.</entry><entry>a_index = 0</entry></row><row><entry>37.</entry><entry>t_index = 1</entry></row><row><entry>38.</entry><entry>g_index = 2</entry></row><row><entry>39.</entry><entry>c_index = 3</entry></row><row><entry>40.</entry><entry>n_index = 4</entry></row><row><entry>41.</entry><entry /></row><row><entry>42.</entry><entry>def get_counts_by_column(base, count, library):</entry></row><row><entry>43.</entry><entry> current_count = library[count]</entry></row><row><entry>44.</entry><entry> if_base == ‘A’:</entry></row><row><entry>45.</entry><entry>  current_count[a_index] += 1</entry></row><row><entry>46.</entry><entry> elif base == ‘T’:</entry></row><row><entry>47.</entry><entry>  current_count[t_index] += 1</entry></row><row><entry>48.</entry><entry> elif base == ‘G’</entry></row><row><entry>49.</entry><entry>  current_count[g_index] += 1</entry></row><row><entry>50.</entry><entry> elif base == ‘C’:</entry></row><row><entry>51.</entry><entry>  current_count[c_index] += 1</entry></row><row><entry>52.</entry><entry> elif base == ‘N’:</entry></row><row><entry>53.</entry><entry>  current_count[n_index] += 1</entry></row><row><entry>54.</entry><entry /></row><row><entry>55.</entry><entry>def dna_counts(list_of_sequences, sample):</entry></row><row><entry>56.</entry><entry> first_oligo = list_of_sequences[0]</entry></row><row><entry>57.</entry><entry> for i in range (len(first_oligo)):</entry></row><row><entry>58.</entry><entry>  sample.append([0,0,0,0,0])</entry></row><row><entry>59.</entry><entry> for j in range(len(first_oligo)):</entry></row><row><entry>60.</entry><entry>  for i in range(len(list_of_sequences)):</entry></row><row><entry>61.</entry><entry>   get_counts_by_column(list_of_sequences[i][j], j, libname)</entry></row><row><entry>62.</entry><entry /></row><row><entry>63.</entry><entry /></row><row><entry>64.</entry><entry>for file in list_of_filenames:</entry></row><row><entry>65.</entry><entry> spacer_list = open(file).read( ).splitlines( )</entry></row><row><entry>66.</entry><entry> output2=[ ]</entry></row><row><entry>67.</entry><entry> dna_counts(spacer_list, output2)</entry></row><row><entry>68.</entry><entry> with open(file + “.csv”, “wb”) as f:</entry></row><row><entry>69.</entry><entry>  writer = csv.writer(f)</entry></row><row><entry>70.</entry><entry>  writer.writerows(output2)</entry></row><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

1002<tables id="TABLE-US-00179" num="00179"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><thead><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Supplementary Note 2:</entry></row><row><entry>Sequences of proteins used in this study</entry></row><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry>Protein sequence of expressed BE3</entry></row><row><entry>(SEQ ID NO: 185)</entry></row><row><entry>MGSSHHHHHHSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYE</entry></row><row><entry></entry></row><row><entry>INWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWS</entry></row><row><entry></entry></row><row><entry>PCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTI</entry></row><row><entry></entry></row><row><entry>QIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPP</entry></row><row><entry></entry></row><row><entry>CLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESA</entry></row><row><entry></entry></row><row><entry>TPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNL</entry></row><row><entry></entry></row><row><entry>IGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSF</entry></row><row><entry></entry></row><row><entry>FHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD</entry></row><row><entry></entry></row><row><entry>KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE</entry></row><row><entry></entry></row><row><entry>ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG</entry></row><row><entry></entry></row><row><entry>LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLS</entry></row><row><entry></entry></row><row><entry>DAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKY</entry></row><row><entry></entry></row><row><entry>KEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED</entry></row><row><entry></entry></row><row><entry>LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFR</entry></row><row><entry></entry></row><row><entry>IPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTN</entry></row><row><entry></entry></row><row><entry>FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKA</entry></row><row><entry></entry></row><row><entry>IVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL</entry></row><row><entry></entry></row><row><entry>LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV</entry></row><row><entry></entry></row><row><entry>MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLI</entry></row><row><entry></entry></row><row><entry>HDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL</entry></row><row><entry></entry></row><row><entry>VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILK</entry></row><row><entry></entry></row><row><entry>EHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFL</entry></row><row><entry></entry></row><row><entry>KDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYVVRQLLNAKLITQRK</entry></row><row><entry></entry></row><row><entry>FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEN</entry></row><row><entry></entry></row><row><entry>DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTA</entry></row><row><entry></entry></row><row><entry>LIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFF</entry></row><row><entry></entry></row><row><entry>KTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVK</entry></row><row><entry></entry></row><row><entry>KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV</entry></row><row><entry></entry></row><row><entry>AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLII</entry></row><row><entry></entry></row><row><entry>KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLK</entry></row><row><entry></entry></row><row><entry>GSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK</entry></row><row><entry></entry></row><row><entry>HRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDAT</entry></row><row><entry></entry></row><row><entry>LIHQSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILML</entry></row><row><entry></entry></row><row><entry>PEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDS</entry></row><row><entry></entry></row><row><entry>NGENKIKMLSGGSPKKKRKV</entry></row><row><entry></entry></row><row><entry>Protein sequence of expressed HF-BE3</entry></row><row><entry>(SEQ ID NO: 186)</entry></row><row><entry>MGSSHHHHHHSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYE</entry></row><row><entry></entry></row><row><entry>INWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWS</entry></row><row><entry></entry></row><row><entry>PCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTI</entry></row><row><entry></entry></row><row><entry>QIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPP</entry></row><row><entry></entry></row><row><entry>CLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESA</entry></row><row><entry></entry></row><row><entry>TPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNL</entry></row><row><entry></entry></row><row><entry>IGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSF</entry></row><row><entry></entry></row><row><entry>FHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD</entry></row><row><entry></entry></row><row><entry>KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE</entry></row><row><entry></entry></row><row><entry>ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG</entry></row><row><entry></entry></row><row><entry>LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLS</entry></row><row><entry></entry></row><row><entry>DAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKY</entry></row><row><entry></entry></row><row><entry>KEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED</entry></row><row><entry></entry></row><row><entry>LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFR</entry></row><row><entry></entry></row><row><entry>IPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTA</entry></row><row><entry></entry></row><row><entry>FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKA</entry></row><row><entry></entry></row><row><entry>IVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL</entry></row><row><entry></entry></row><row><entry>LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV</entry></row><row><entry></entry></row><row><entry>MKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMALI</entry></row><row><entry></entry></row><row><entry>HDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL</entry></row><row><entry></entry></row><row><entry>VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILK</entry></row><row><entry></entry></row><row><entry>EHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFL</entry></row><row><entry></entry></row><row><entry>KDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYVVRQLLNAKLITQRK</entry></row><row><entry></entry></row><row><entry>FDNLTKAERGGLSELDKAGFIKRQLVETRAITKHVAQILDSRMNTKYDEN</entry></row><row><entry></entry></row><row><entry>DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTA</entry></row><row><entry></entry></row><row><entry>LIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFF</entry></row><row><entry></entry></row><row><entry>KTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVK</entry></row><row><entry></entry></row><row><entry>KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV</entry></row><row><entry></entry></row><row><entry>AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLII</entry></row><row><entry></entry></row><row><entry>KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLK</entry></row><row><entry></entry></row><row><entry>GSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK</entry></row><row><entry></entry></row><row><entry>HRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDAT</entry></row><row><entry></entry></row><row><entry>LIHQSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILML</entry></row><row><entry></entry></row><row><entry>PEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDS</entry></row><row><entry></entry></row><row><entry>NGENKIKMLSGGSPKKKRKV</entry></row><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

1003<tables id="TABLE-US-00180" num="00180"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="399pt" align="left" /><thead><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Supplementary Note 3: Sequences of oligonucleotides used in the present study</entry></row><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry>Unpublished Primers used to amplify off target genomic DNA for HTS in human cells</entry></row><row><entry>(SEQ ID NOS: 506-513)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="154pt" align="left" /><colspec colname="2" colwidth="245pt" align="left" /><tbody valign="top"><row><entry>fwd_VEGFA_site2_off_target_1_human</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCTACAAGTAACAGTCCAA</entry></row><row><entry></entry></row><row><entry /><entry>GAA</entry></row><row><entry></entry></row><row><entry>rev_VEGFA_site2_off_target_1_human</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTTCTGCAACTTAACTTACGTGAAA</entry></row><row><entry></entry></row><row><entry>fwd_VEGFA_site2_off_target_2_human</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNACCAAGCCCATTTGTCCAGG</entry></row><row><entry></entry></row><row><entry>rev_VEGFA_site2_off_target_2_human</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTTCTTTTTGAGCTTTGGGC</entry></row><row><entry></entry></row><row><entry>fwd_VEGFA_site2_off_target_3_human</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCATACCAGCAGCAGTTCC</entry></row><row><entry></entry></row><row><entry>rev_VEGFA_site2_off_target_3_human</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCTCACCTCAGCTCCTGCAC</entry></row><row><entry></entry></row><row><entry>fwd_VEGFA_site2_off_target_4_human</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCCACTGATTCTACACCATG</entry></row><row><entry></entry></row><row><entry /><entry>GT</entry></row><row><entry></entry></row><row><entry>rev_VEGFA_site2_off_target_4_human</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGGAGTTCCCAACCTTTTTGACA</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="399pt" align="left" /><tbody valign="top"><row><entry>Other primers (for off target sites associated with HEK_3, EMX1, FANCF) were previously</entry></row><row><entry>published</entry></row><row><entry></entry></row><row><entry>Primers used to amplify off target genomic DNA for HTS in murine cells</entry></row><row><entry>(SEQ ID NOS: 514-521)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="154pt" align="left" /><colspec colname="2" colwidth="245pt" align="left" /><tbody valign="top"><row><entry>fwd_VEGFA_site2_off_target_1_murine</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTGGCTGGAGATTCAGAGAC</entry></row><row><entry></entry></row><row><entry /><entry>AC</entry></row><row><entry></entry></row><row><entry>rev_VEGFA_site2_off_target_1_murine</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGCCCCTTCTGACACACATAC</entry></row><row><entry></entry></row><row><entry>fwd_VEGFA_site2_off_target_2_murine</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNACCCCTCAAGGCTTGACATT</entry></row><row><entry></entry></row><row><entry /><entry>TC</entry></row><row><entry></entry></row><row><entry>rev_VEGFA_site2_off_target_2_murine</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTGAAAAGTTGGGAGAGGGGATG</entry></row><row><entry></entry></row><row><entry>fwd_VEGFA_site2_off_target_3_murine</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTTGTACCCCAGTCCCCTCA</entry></row><row><entry></entry></row><row><entry /><entry>TC</entry></row><row><entry></entry></row><row><entry>rev_VEGFA_site2_off_target_3_murine</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTGAAGTTACGGGGATGTCACTTG</entry></row><row><entry></entry></row><row><entry>fwd_VEGFA_site2_off_target_4_murine</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTAACATCCAGTCTCCCAAA</entry></row><row><entry></entry></row><row><entry /><entry>CACA</entry></row><row><entry></entry></row><row><entry>rev_VEGFA_site2_off_target_4_murine</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTACACACACACACTACTAGGACA</entry></row><row><entry></entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="399pt" align="left" /><tbody valign="top"><row><entry>Primers used to amplify on target genomic DNA for HTS in murine cells</entry></row><row><entry>(SEQ ID NOS: 522-523)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="154pt" align="left" /><colspec colname="2" colwidth="245pt" align="left" /><tbody valign="top"><row><entry>fwd_VEGFA_site2_on_target_murine</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCGCTACTACGGAGCGAGAAG</entry></row><row><entry></entry></row><row><entry>rev_VEGFA_site2_on_target_murine</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTACAGGGGCAAAGTGAGTGAC</entry></row><row><entry></entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="399pt" align="left" /><tbody valign="top"><row><entry>Primers used for generating PCR products to serve as substrates for T7 transcription of sgRNAs</entry></row><row><entry>(SEQ ID NOS: 524-529)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="154pt" align="left" /><colspec colname="2" colwidth="245pt" align="left" /><tbody valign="top"><row><entry>rev_sgRNA_T7: used in all cases</entry><entry>AAAAAAAGCACCGACTCGGTGCCAC</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_EMX1</entry><entry>TAATACGACTCACTATAGGGAGTCCGAGCAGAAGAAGAAGTTTTAGAGCTAGAAATA</entry></row><row><entry></entry></row><row><entry /><entry>GCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_FANCF</entry><entry>TAATACGACTCACTATAGGGGAATCCCTTCTGCAGCACCGTTTTAGAGCTAGAAATA</entry></row><row><entry></entry></row><row><entry /><entry>GCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_HEK_site_3</entry><entry>TAATACGACTCACTATAGGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATA</entry></row><row><entry></entry></row><row><entry /><entry>GCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_VEGFA_site_2</entry><entry>TAATACGACTCACTATAG GACCCCCTCCACCCCGCCTCGTTTTAGAGCTAGAAATA</entry></row><row><entry></entry></row><row><entry /><entry>GCA</entry></row><row><entry></entry></row><row><entry>fwd_sgRNA_T7_TC_repeat_in_vitro</entry><entry>TAATACGACTCACTATAGGTCTCTCTCTCTCTCTCTCTCGTTTTAGAGCTAGAAATA</entry></row><row><entry></entry></row><row><entry /><entry>GCA</entry></row><row><entry></entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="399pt" align="left" /><tbody valign="top"><row><entry>Primers used for generating sgRNA transfection glasmids</entry></row><row><entry>The pFYF1320 plasmid was used as template as previously described (Komor et al). The sequence</entry></row><row><entry>of other sgRNA plasmids was previously reported</entry></row><row><entry>(SEQ ID NOS: 530-531)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="154pt" align="left" /><colspec colname="2" colwidth="245pt" align="left" /><tbody valign="top"><row><entry>rev_sgRNA_plasmid</entry><entry>GGTGTTTCGTCCTTTCCACAAG</entry></row><row><entry></entry></row><row><entry>fwd_VEGFA_site_2</entry><entry>GACCCCCTCCACCCCGCCTCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC</entry></row><row><entry></entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="399pt" align="left" /><tbody valign="top"><row><entry>Sequences of ssDNA substrates used in in vitro deaminase assays</entry></row><row><entry>(SEQ ID NOS: 532-533)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="154pt" align="left" /><colspec colname="2" colwidth="245pt" align="left" /><tbody valign="top"><row><entry>fwd_TC_repeat_substrate</entry><entry>ACGTAAACGGCCACAAGTTCGCGGCGATCGTCTCTCTCTCTCTCTCTCTCTGGATCG</entry></row><row><entry></entry></row><row><entry /><entry>CCTGGCATCTTCTTCAAGGACG</entry></row><row><entry></entry></row><row><entry>rev_TC_repeat_substrate</entry><entry>CGTCCTTGAAGAAGATGCCAGGCGATCCAGAGAGAGAGAGAGAGAGAGACGATCGCC</entry></row><row><entry></entry></row><row><entry /><entry>GCGAACTTGTGGCCGTTTACGT</entry></row><row><entry></entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="399pt" align="left" /><tbody valign="top"><row><entry>Previously published primers used to amplify off target genomic DNA for HTS in human cells</entry></row><row><entry>(SEQ ID NOS: 534-557)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="154pt" align="left" /><colspec colname="2" colwidth="245pt" align="left" /><tbody valign="top"><row><entry>fwd_EMX1_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCAGCTCAGCCTGAGTGTTGA</entry></row><row><entry></entry></row><row><entry>rev_EMX1_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCTCGTGGGTTTGTGGTTGC</entry></row><row><entry></entry></row><row><entry>fwd_FANCF_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCATTGCAGAGAGGCGTATCA</entry></row><row><entry></entry></row><row><entry>rev_FANCF_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGGGGTCCCAGGTGCTGAC</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site3_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNATGTGGGCTGCCTAGAAAGG</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site3_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCCCAGCCAAACTTGTCAACC</entry></row><row><entry></entry></row><row><entry>fwd_EMX1_off1_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAGTAGCCTCTTTCTCAATGT</entry></row><row><entry></entry></row><row><entry /><entry>GC</entry></row><row><entry></entry></row><row><entry>rev_EMX1_off1_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGCTTTCACAAGGATGCAGTCT</entry></row><row><entry></entry></row><row><entry>fwd_EMX1_off2_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGAGCTAGACTCCGAGGGGA</entry></row><row><entry></entry></row><row><entry>rev_EMX1_off2_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTCGTCCTGCTCTCACTT</entry></row><row><entry></entry></row><row><entry>fwd_EMX1_0ff3_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAGAGGCTGAAGAGGAAGACCA</entry></row><row><entry></entry></row><row><entry>rev_EMX1_off3_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGGCCCAGCTGTGCATTCTAT</entry></row><row><entry></entry></row><row><entry>fwd_FANCF_off1_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAACCCACTGAAGAAGCAGGG</entry></row><row><entry></entry></row><row><entry>rev_FANCF_off1_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGGTGCTTAATCCGGCTCCAT</entry></row><row><entry></entry></row><row><entry>fwd_FANCF_0ff2_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCAGTGTTTCCATCCCGAA</entry></row><row><entry></entry></row><row><entry>rev_FANCF_off2_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCCTCTGACCTCCACAACTCT</entry></row><row><entry></entry></row><row><entry>fwd_FANCF_off3_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTGGGTACAGTTCTGCGTGT</entry></row><row><entry></entry></row><row><entry>rev_FANCF_off3_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTCACTCTGAGCATCGCCAAG</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site3_offl_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCCCTGTTGACCTGGAGAA</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site3_offl_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCACTGTACTTGCCCTGACCA</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site3_off2_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTGGTGTTGACAGGGAGCAA</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site3_off2_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGAGATGTGGGCAGAAGGG</entry></row><row><entry></entry></row><row><entry>fwd_HEK293_site3_off3_HTS</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGAGAGGGAACAGAAGGGCT</entry></row><row><entry></entry></row><row><entry>rev_HEK293_site3_off3_HTS</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCCAAAGGCCCAAGAACCT</entry></row><row><entry></entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="399pt" align="left" /><tbody valign="top"><row><entry>Primers used to amplify on target genomic DNA for HTS in zebrafish</entry></row><row><entry>(SEQ ID NOS: 558-563)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="154pt" align="left" /><colspec colname="2" colwidth="245pt" align="left" /><tbody valign="top"><row><entry>fwd_TYR1_zebrafish</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTTCCCCCGAGTCTGCACCT</entry></row><row><entry></entry></row><row><entry>rev_TYR1_zebrafish</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCGAACTTGCATTCGCCGCAA</entry></row><row><entry></entry></row><row><entry>fwd_TYR2_zebrafish</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTCTGCCTTGGCATCGGGTG</entry></row><row><entry></entry></row><row><entry>rev_TYR2_zebrafish</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTCACCATACCGCCCCTAGAACTAACATTC</entry></row><row><entry></entry></row><row><entry>fwd_TYR3_zebrafish</entry><entry>ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNACAACTGCTTTCCATGGTGT</entry></row><row><entry></entry></row><row><entry /><entry>GT</entry></row><row><entry></entry></row><row><entry>rev_TYR3_zebrafish</entry><entry>TGGAGTTCAGACGTGTGCTCTTCCGATCTTCCCAGGGCTTTCGTGGAGA</entry></row><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="5"><colspec colname="1" colwidth="70pt" align="left" /><colspec colname="2" colwidth="70pt" align="left" /><colspec colname="3" colwidth="147pt" align="left" /><colspec colname="4" colwidth="56pt" align="center" /><colspec colname="5" colwidth="56pt" align="left" /><tbody valign="top"><row><entry /><entry>Site</entry><entry>Sequence</entry><entry>SEQ ID NO</entry><entry /></row><row><entry namest="1" nameend="5" align="center" rowsep="1" /></row><row><entry /><entry>TYR1</entry><entry>GTC₃AGGTC₈GAGGGTTCTGTCAGG</entry><entry>564</entry><entry /></row><row><entry></entry></row><row><entry /><entry>TYR2</entry><entry>CTTC₄C₅AGGATGAGAACACAGAGG</entry><entry>565</entry><entry /></row><row><entry></entry></row><row><entry /><entry>TYR3</entry><entry>CAAC₄C₅AC₇TGCTCAAAGATGCTGG</entry><entry>566</entry></row><row><entry namest="1" nameend="5" align="center" rowsep="1" /></row></tbody></tgroup></table></tables><br /> Supplementary Table 1. Protospacer and PAM Sequences for the Zebrafish Genomic Loci Studied in this Work.

Example 17: C: G-to-T:A Base Editors with Higher Efficiency and Product Purity

1004Base editing is the programmable conversion of target C:G base pairs to T:A base pairs without inducing double-stranded DNA breaks or requiring homology-directed repair using engineered fusions of Cas9 variants and cytidine deaminases (1). The third-generation base editor (BE3) and related technologies have been successfully used by many researchers in a wide range of organisms (2-13). The product distribution of base editing—the frequency with which the target C:G base pair is converted to mixtures of undesired byproducts, along with the desired T:A product-varies in a target site-dependent manner (2, 3, 6-8). Here we characterize determinants of base editing outcomes in human cells, and establish that the formation of undesired products is dependent on uracil N-glycosylase (UNG), and is more likely to occur at target sites containing only a single C within the base editing activity window. The constructs CDA1-BE3 and AID-BE3, which use cytidine deaminase homologs that increase base editing efficiency for some sequences, were engineered. Additionally, a fourth-generation <i>S. pyogenes </i>Cas9-derived base editor (BE4) that more efficiently blocks access of UNG to base-edited intermediates was also engineered. Compared with BE3, BE4 increases by approximately 50% the efficiency of C:G to T:A base editing, while halving the frequency of undesired byproducts. These improvements were also applied to yield a <i>S. aureus </i>Cas9-derived BE4 (SaBE4), which is substantially smaller than BE4 and has an alternative targeting scope.

0000Introduction

1005Traditional genome editing methods introduce a double-stranded DNA break (DSB) at a genomic target locus (14). The cellular response to a DSB lesion primarily proceeds through nonhomologous end joining (NHEJ) and related processes (15). Although NHEJ usually rejoins the two ends flanking the DSB, under typical genome editing conditions DSBs are continuously reintroduced, eventually resulting in the accumulation of insertions and deletions (indels) or translocations at the site of the DSB and disruption of the corresponding genomic locus (16). Actively dividing cells can also respond to DSBs by initiating homology-directed repair (HDR) in the presence of a donor DNA template containing homology to the regions surrounding the DSB, which allows researchers to more precisely and predictably manipulate genomes than is possible through NHEJ (17). HDR-dependent genome editing is limited by low efficiency arising from competition with NHEJ outcomes, and from the dependence of HDR on mitosis (18).

1006The development of base editing, which enables the direct, irreversible conversion of a C:G base pair to a T:A base pair in a programmable manner without requiring HDR or the introduction of a DSB, has been reported (1). Base editors consist of a single-stranded DNA-specific cytidine deaminase enzyme tethered to a catalytically impaired Cas9 protein and a base excision repair inhibitor (1, 4, 9, 10). The Cas9 variant binds a genomic locus of interest, programmed by a corresponding guide RNA. Formation of the protein:RNA:DNA ternary “R-loop” complex (19) exposes a small (˜5-nt) window of single-stranded DNA that serves as a substrate for the tethered cytidine deaminase enzyme. Any cytidines within this window are hydrolytically deaminated to uracils, resulting in G:U intermediates.

1007Base excision repair (BER) is the cell's primary response to G:U mismatches and is initiated by excision of the uracil by uracil N-glycosylase (UNG)(20). In an effort to protect the edited G:U intermediate from excision by UNG, an 83-amino acid uracil glycosylase inhibitor (UGI) was fused directly to the C-terminus of catalytically dead Cas9 (dCas9) (1). To manipulate cellular DNA mismatch repair systems into preferentially replacing the G in the G:U mismatch with an A, the Ala 840 amino acid in dCas9 was reverted to His, enabling the Cas9 protein to nick the DNA strand opposite the newly formed uracil, resulting in much more efficient conversion of the G:U intermediate to desired A:U and A:T products (1). Combining these two engineering efforts resulted in BE3, a single protein consisting of a three-part fusion of the APOBEC1 cytidine deaminase enzyme tethered through a 16-amino acid linker to <i>S. pyogenes </i>dCas9(A840H), which is covalently linked to UGI through a 4-amino acid linker (1). BE3 and related base editors have now been employed for a wide variety of applications including plant genome editing, in vivo mammalian genome editing, targeted mutagenesis, and knockout studies (2-13). The scope of base editing has been recently expanded by reporting BE3 variants with altered PAM requirements (4), narrowed editing windows (4), reduced off-target editing (10), and small molecule dependence (21).

1008At some loci, base editors such as BE3 give rise to undesired byproducts in which the target C:G base pair is converted into a G:C or A:T base pair, rather than the desired T:A product (2, 3, 6-8). Here we illuminate determinants of base editing product purity, and establish that UNG activity is required for the formation of undesired byproducts. It has been determined that blocking UNG access to the uracil intermediate is especially crucial for target loci in which a single C is within the editing window in order to minimize undesired products. Fourth-generation base editors, BE4 and SaBE4, that perform base editing with higher efficiency and greatly improved product purity compared to previously described base editors including BE3 were engineered.

0000Results

0000UNG Activity is Required for Byproduct Formation

1009Undesired base editing byproducts may arise during base excision repair due to the formation and error-prone resolution of abasic sites within the uracil-containing DNA strand. To determine if the product purity of base editing in cells lacking uracile N-glycosylase (UNG) improves, HAP1 cells (a haploid human cell line) and HAP1 UNG⁻ cells were nucelofected with plasmids encoding BE3 and sgRNAs targeting the EMX1, FANCF, HEK2, HEK3, HEK4, or RNF2 loci (see <figref idref="DRAWINGS">FIG. 135B</figref> for target sequences). Three days post-nucleofection, genomic DNA was extracted and the target loci were amplified by PCR and analyzed by high-throughput DNA sequencing (HTS). Base editing product purity is defined as the percent of edited sequencing reads (reads in which the target C has been converted to A, G, or T) in which the target C is edited to a T. The base editing product purity of BE3-treated HAP1 cells averaged 68±6% (mean±S.D. for n=3 biological replicates) across 12 target Cs in the six loci. In HAP1 UNG cells, all 12 target Cs tested were base edited with product purities >98% (<figref idref="DRAWINGS">FIG. 135A</figref>). In addition, indel frequencies at all six tested loci decreased 7- to 100-fold upon UNG knockout (<figref idref="DRAWINGS">FIG. 135C</figref>). These data strongly implicate UNG activity as necessary for undesired product formation during base editing, consistent with a model in which abasic site formation and subsequent base excision repair with error-prone polymerases leads to randomization of the target nucleotide and occasional strand breaks that result in indels.

0000Targets with Multiple Editable Cs Exhibit Higher Product Purity

1010Base editing efficiency by BE3 can be lower for some (but not all) target Cs that are immediately downstream of a G (1), consistent with the known sequence preference of APOBEC1 (22) (<figref idref="DRAWINGS">FIG. 136A</figref>). In an effort to efficiently edit such targets, BE3 variants in which replaced the APOBEC1 deaminase was replaced with CDA1 (to generate CDA1-BE3), AID (to generate AID-BE3), or APOBEC3G (to generate APOBEC3G-BE3), three single-stranded DNA-specific cytidine deaminase enzymes with different sequence preferences, were generated (23). HEK293T cells were transfected with plasmids encoding these BE3 variants and sgRNAs targeting the EMX1, FANCF, HEK2, HEK3, HEK4, or RNF2 loci. Three days post-transfection, genomic DNA was extracted and the target loci were amplified by PCR and assessed for base editing using HTS. More efficient editing of target Cs that immediately follow a G was observed with CDA1-BE3 and AID-BE3 compared to BE3 (<figref idref="DRAWINGS">FIG. 136A</figref>, <figref idref="DRAWINGS">FIGS. 140A-D</figref>, and <figref idref="DRAWINGS">FIGS. 146-151</figref>). In general, CDA1-BE3 and AID-BE3 exhibited lower editing efficiencies than BE3 at target Cs that do not follow a G (<figref idref="DRAWINGS">FIGS. 140A-D</figref>). In contrast, APOBEC3G-BE3 exhibited unpredictable sequence preferences, with overall lower yields of C-to-T editing compared to BE3. These findings suggest that CDA1-BE3 and AID-BE3 may offer higher editing efficiencies over BE3 for some target 5′-GC-3′ sequences.

1011While analyzing these data, it was noted that the product purities of CDA1-BE3 and AID-BE3 were typically higher than those of BE3 at those sites for which CDA1-BE3 and AID-BE3 edited more Cs than BE3 (<figref idref="DRAWINGS">FIGS. 136A-D</figref>). For example, at the HEK4 locus, BE3 edits only a single C efficiently (the C not preceded by a G) but both CDA1-BE3 and AID-BE3 edit three Cs (<figref idref="DRAWINGS">FIGS. 140A-C</figref>). The product purity of BE3 at this locus is 50±7% (mean±S.D. for n=3 biological replicates), while the product purity of CDA1-BE3 and AID-BE3 are 97±2% and 93±2%, respectively. Moreover, EMX1 and FANCF, edited by BE3 with product purities of 84±3% and 91±2%, respectively, contain multiple Cs that are edited with comparable efficiency (Figure S2), while HEK2 and RNF2, edited by BE3 with much lower product purities of 28±3% and 64±3%, respectively, contain multiple Cs that are edited with unequal efficiencies (<figref idref="DRAWINGS">FIGS. 141A-C</figref>). CDA1-BE3 and AID-BE3, which edit both Cs within the HEK2 locus with comparable efficiencies, exhibit much higher product purities at this locus (85±5% and 81±4%, respectively) (<figref idref="DRAWINGS">FIGS. 136A-D</figref> and <figref idref="DRAWINGS">FIG. 140C</figref>). The possibility that at the HEK2 and RNF2 sites the multiple Cs are initially converted to Us by BE3 with comparable efficiency and then processed with different efficiencies by DNA repair systems was ruled out. Given this, similar product distributions would be expected when these sites were treated with BE3 versus CDA1-BE3 or AID-BE3, rather than the different product distributions observed (<figref idref="DRAWINGS">FIG. 136B</figref> and <figref idref="DRAWINGS">FIGS. 146-151</figref>). Instead, an isolated G:U may be more readily processed by UNG than clusters of G:U lesions. It is possible that the processivity of the cytidine deaminase domain in BE3 (1, 24) may increase the residence time of BE3 at loci containing multiple editable Cs, thereby blocking access by UNG more effectively than at loci containing a single editable C.

1012The relationship between product purity, the number of edited Cs in individual sequencing reads, and UNG activity was further analyzed. To reveal the fate of base edited DNA in the absence of explicit UNG inhibition, the UGI component of BE3 was removed to generate BE3B. HEK293T cells were transfected with plasmids encoding BE3 or BE3B and sgRNAs targeting the EMX1, FANCF, HEK2, HEK3, HEK4, or RNF2 loci. As expected given the role of UNG in diversifying base editing outcomes established above, the product purities at all target Cs greatly decreased in BE3B-treated DNA compared with BE3-treated DNA, with the fraction of editing products containing non-Ts increasing by an average of 1.8±0.4-fold (<figref idref="DRAWINGS">FIG. 142B</figref>).

1013Individual DNA sequencing reads from HEK293T cells treated with sgRNAs targeting the multi-C sites HEK2, HEK3, and RNF2 and either BE3 or BE3B were analyzed. For each site, the primary target C was designated as the nucleotide modified most efficiently. Across all three sites, an average of 80±10% of sequencing reads that contained an undesired C to non-T edit of the primary target C exhibited only that single base editing event (<figref idref="DRAWINGS">FIGS. 142A-D</figref> and <figref idref="DRAWINGS">FIG. 143</figref>). In contrast, across the same three multi-C sites, a much lower average of 32±4% of sequencing reads containing a clean C-to-T edit of the primary target C exhibited only that single clean base editing event (<figref idref="DRAWINGS">FIGS. 142A-D</figref> and <figref idref="DRAWINGS">FIG. 143</figref>). In addition, the distribution of products for BE3B-treated HEK4 DNA, a site that contains only one C within the editing window, roughly follows the ratio of 1:3:1 for A:G:T (<figref idref="DRAWINGS">FIG. 143D</figref>). These observations collectively indicate that when a single cytidine in a given target is converted to U in the absence of UGI, it is processed efficiently by UNG-initiated BER to give a mixture of products.

1014These data are consistent with a model in which clustered G:U mismatches are processed differently than isolated G:U mismatches, and are more likely to produce clean C-to-T edits. When only a single C-to-T editing event is desired, the above observations suggest that UNG inhibition is critical to minimize undesired byproducts. However, when performing targeted random mutagenesis using dCas9-deaminase fusions, such as with TAM(8) and CRISPR-X(2), the above observations suggest that target sites with only a single editable C will maximize product mixtures.

0000Optimization of BE3 Architecture for Improved Product Purity

1015The UGI component of BE3 was replaced with a single-stranded DNA binding (SSB) protein to yield SSB-BE3, such that SSB may block the uracil-containing ssDNA portion of the R-loop from being accessed by UNG. Large decreases in base editing efficiency by SSB-BE3 were observed, with all seven Cs across the four sites exhibiting an average of only 1.9±0.5% C-to-T conversion (<figref idref="DRAWINGS">FIG. 137C</figref>).

1016Since the relative positioning of APOBEC, UGI, and UNG during steps that determine base editing outcomes are not known, UGI was relocated to the N-terminus of BE3 (N-UGI-BE3) in an effort to improve UNG inhibition. Moving UGI to the N-terminus of BE3 resulted in an average decrease in C-to-T editing percentages across all seven tested target Cs of 2.3±0.6-fold compared to BE3 (<figref idref="DRAWINGS">FIG. 137C</figref>), and a decrease in overall product purity at all four sites compared to BE3 averaging 2.2±0.5-fold (<figref idref="DRAWINGS">FIG. 137B</figref>).

1017In contrast, appending an additional copy of UGI to the C-terminus of BE3 (BE3-2×UGI) resulted in large increases in product purities relative to BE3 and C-to-T editing percentages comparable to those of BE3. Non-T editing products decreased an average of 2.2±0.8-fold across the four loci tested (<figref idref="DRAWINGS">FIG. 137B</figref>). These observations suggest that addition of a second copy of UGI substantially decreases the access of UNG to the G:U base editing intermediate, thereby greatly improving product purity.

1018Because the above experiments also revealed the sensitivity of base editing outcomes to the architecture of the components, next we optimized the linkers between BE3 components to further increase product purities and editing efficiencies. We varied the rAPOBEC1-dCas9(A840H) linker from 16 amino acids (BE3) to 32 amino acids (BE3C) and the dCas9(A840H)-UGI linker from 4 (BE3) to 9 (BE3D) to 16 amino acids (BE3E, <figref idref="DRAWINGS">FIG. 138A</figref>). Non-T product formation on average decreased 1.3±0.1-fold when the dCas9(A840H)-UGI linker was nine amino acid residues in length (BE3D) instead of four amino acids (BE3) (<figref idref="DRAWINGS">FIG. 138D</figref>), with no apparent differences in C-to-T editing efficiencies (<figref idref="DRAWINGS">FIG. 138C</figref>). Increasing the rAPOBEC1-dCas9(A840H) linker from 16 amino acids (BE3) to 32 amino acids (BE3C) elevated C-to-T editing efficiencies an average of 1.2±0.1-fold at the HEK2 locus (<figref idref="DRAWINGS">FIG. 138C</figref>). This locus was previously the most unevenly edited multi-C site tested (<figref idref="DRAWINGS">FIGS. 141A-C</figref>), and extending this linker led to a reduction in preferential editing of C6 over C4 (the ratio of the percentage of sequencing reads that are edited at C6 to that of C4) from 2.6±0.2-fold to 1.8±0.1-fold. We reasoned that this longer linker may allow the deaminase better access to the ssDNA in the R-loop and result in more uniform deamination when multiple target Cs are present in the base editing window. BE3C also exhibited comparable or improved base editing efficiencies and product purities at the other loci tested (<figref idref="DRAWINGS">FIGS. 138C-D</figref>).

0000BE4, a C:G to T:a Base Editor with Enhanced Efficiency and Product Purity

1019The base editor construct BE4 was engineered by combining all three improvements—extending the rAPOBEC1-dCas9 linker to 32 amino acids, extending the dCas9-UGI linker to 9 amino acids, and appending a second copy of UGI to the C-terminus of the construct with another 9-amino acid linker. Target-AID, an alternative base editor construct reported by Nishida et. al. (9), was also cloned into the same plasmid backbone as BE4. HEK293T cells were transfected with plasmids encoding BE3, BE4, or Target-AID and sgRNAs targeting the EMX1, FANCF, HEK2, HEK3, HEK4, or RNF2 loci. Three days post transfection, genomic DNA was extracted and the target loci were amplified by PCR and analyzed by HTS. An average increase in C-to-T editing efficiencies of 1.5±0.3-fold across all twelve edited Cs for BE4 relative to BE3 was observed (<figref idref="DRAWINGS">FIG. 139C</figref>). Although the average efficiency of C-to-T editing for Target-AID at the same positions analyzed was 1.5±0.5-fold lower than that of BE3 and 2.1±0.5-fold lower than that of BE4, it is important to note that Target-AID, which uses the CDA1 deaminase, appears to have an editing window shifted relative to BE3 and BE4, with optimal editing around positions C3 and C4 (<figref idref="DRAWINGS">FIG. 139C</figref>). This shifted editing window makes comparisons of efficiency and product purity between Target-AID and BE3 or BE4 difficult because a given target C could lie in more optimal or less optimal position within the different editing windows, even when using the same guide RNA.

1020In addition to greater C-to-T editing efficiency, BE4 also exhibited substantially improved product purities relative to BE3 at all genomic loci tested, with an average decrease in non-T product formation of 2.3±0.3-fold (<figref idref="DRAWINGS">FIG. 139D</figref>). As expected from further impeding base excision repair, which can lead to indels (25), decreases in indel rates averaging 2.3±1.1-fold across all six loci following BE4 treatment compared to BE3 were also observed (<figref idref="DRAWINGS">FIGS. 144A-C</figref>). Taken together, these results indicate that BE4 offers high efficiencies of C-to-T editing, high product purities, and low indel formation rates at all loci tested.

1021The BE4 improvements were integrated with <i>S. aureus </i>Cas9 (26) to generate SaBE4, which replaces the <i>S. pyogenes </i>dCas9(A840H) with the smaller <i>S. aureus </i>dCas9(A580N) and can access different targets due to its alternative PAM requirements. HEK293T cells were transfected with plasmids encoding SaBE3 (4) or SaBE4 and sgRNAs targeting the FANCF, HEK3, or HEK4 loci. Consistent with the results comparing BE4 and BE3, we observed an average increase in C-to-T editing efficiencies of 1.4±0.2-fold across all ten edited Cs for SaBE4 relative to SaBE3 (<figref idref="DRAWINGS">FIG. 145A</figref>), with a 1.8±0.5-fold average decrease in undesired non-T editing products (<figref idref="DRAWINGS">FIG. 145B</figref>). These results indicate that the gains in base editing efficiency and product purity that arise from the BE4 enhancements also apply to base editors derived from other Cas9 homologs.

0000Materials and Methods

0000Cloning of Plasmids

1022All plasmids in this study were generated by USER cloning using Phusion U Hot Start polymerase (Thermo Fisher). Deaminase and SSB genes were synthesized as gBlocks Gene Fragments (Integrated DNA Technologies), and Target-AID was obtained from Addgene (plasmid #79620). Protein sequences are listed in the Supplementary Notes.

0000Cell Culture

1023HEK293T (ATCC CRL-3216) cells were maintained in Dulbecco's Modified Eagle's Medium plus GlutaMax (ThermoFisher) supplemented with 10% (v/v) fetal bovine serum (FBS), at 37° C. with 5% CO₂. HAP1 (Horizon Discovery C631) and HAP1 UNG (Horizon Discovery HZGHC001531c012) were maintained in Iscove's Modified Dulbecco's Medium plus GlutaMax (ThermoFisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS), at 37° C. with 5% CO₂.

0000Transfections

1024HEK293T cells were seeded on 48-well collagen-coated BioCoat plates (Corning) and transfected at approximately 75% confluency. Briefly, 750 ng of BE and 250 ng of sgRNA expression plasmids were transfected using 1.5 μL of LIPOFECTAMINE® 2000 transfection reagent (ThermoFisher Scientific) per well according to the manufacturer's protocol.

1025HAP1 and HAP1 UNG⁻ cells were nucleofected using the SE Cell Line 4DNucleofector™ X Kit S (Lonza) according to the manufacturer's protocol. Briefly, 4×10⁵cells were nucleofected with 300 ng of BE and 100 ng of sgRNA expression plasmids using the 4DNucleofector™ program DZ-113.

0000High-Throughput DNA Sequencing of Genomic DNA Samples

1026Transfected cells were harvested after 3 days and the genomic DNA was isolated by incubating cells in lysis buffer (10 mM Tris-HCl pH 8.0, 0.05% SDS, 25 μg/mL proteinase K) at 37° C. for 1 hr followed by 80° C. for 30 min. Genomic regions of interest were amplified by PCR with flanking HTS primer pairs as previously described (6, 1). PCR amplification was carried out with Phusion high-fidelity DNA polymerase (ThermoFisher) according to the manufacturer's instructions and as previously described. Purified DNA was amplified by PCR with primers containing sequencing adaptors. The products were gel-purified and quantified using the QuantiT™ PicoGreen dsDNA Assay Kit (ThermoFisher) and KAPA Library Quantification Kit-Illumina (KAPA Biosystems). Samples were sequenced on an Illumina MiSeq as previously described.

0000Data Analysis

1027Sequencing reads were automatically demultiplexed using MiSeq Reporter (Illumina), and individual FASTQ files were analyzed with a custom Matlab script as previously described (1). Each read was pairwise aligned to the appropriate reference sequence using the Smith-Waterman algorithm. Base calls with a Q-score below 31 were replaced with Ns and were thus excluded in calculating nucleotide frequencies. This treatment yields an expected MiSeq base-calling error rate of approximately 1 in 1,000. Aligned sequences in which the read and reference sequence contained no gaps were stored in an alignment table from which base frequencies could be tabulated for each locus.

1028Indel frequencies were quantified with the previously described Matlab script (5, 6, 1). Briefly, sequencing reads were scanned for exact matches to two 10-bp sequences that flank both sides of a window in which indels might occur. If no exact matches were located, the read was excluded from analysis. If the length of this indel window exactly matched the reference sequence the read was classified as not containing an indel. If the indel window was two or more bases longer or shorter than the reference sequence, then the sequencing read was classified as an insertion or deletion, respectively.

1029In order to evaluate interdependency (linkage disequilibrium) between the base editing outcomes at the multiple target cytidines within an editing window, target site sequences from BE treated cells were analyzed by a custom Python script (Supplementary Note 1). Briefly, sequencing reads were scanned for exact matches to two 7-bp sequences that flank each side of the protospacer. If the intervening region was not exactly 20-bp, then it was excluded further analysis. The protospacer sequences were further filtered into four groups based upon the identity of the nucleotide at the position with the most non-T editing outcomes (the primary target C). For each of these four groups as well as the entire pool, the nucleotide abundance at each of the 20 positions within the protospacer were tallied.

Example 18: Base Editors Comprising an LbCpf1 (Nuclease Dead, Nuclease Active, and Nickase)

1030As discussed above, nucleic acid programmable DNA binding proteins (napDNAbp) of any of the fusion proteins provided herein may be an LbCpf1 protein. In some embodiments, the LbCpf1 protein is nuclease inactive, nuclease active, or an LbCpf1 nickase. Several constructs of fusion proteins comprising forms of LbCpf1 were tested for their ability to make C to T edits in different target sequences. A schematic representation of the constructs tested is shown in <figref idref="DRAWINGS">FIG. 152</figref>. Construct 10 has a domain arrangement of [Apobec]-[LbCpf1]-[UGI]-[UGI]; construct 11 has a domain arrangement of [Apobec]-[LbCpf1]-[UGI]; construct 12 has a domain arrangement of [UGI]-[Apobec]-[LbCpf1]; construct 13 has a domain arrangement of [Apobec]-[UGI]-[LbCpf1]; construct 14 has a domain arrangement of [LbCpf1]-[UGI]-[Apobec]; construct 15 has a domain arrangement of [LbCpf1]-[Apobec]-[UGI]. For each construct three different LbCpf1 proteins were used (D/N/A, which refers to nuclease dead LbCpf1 (D); LbCpf1 nickase (N) and nuclease active LbCpf1 (A)). For each of these constructs, the linkers linking the domains are shown below, where XTEN refers to the XTEN linker having the sequence SGSETPGTSESATPES (SEQ ID NO: 604), and BPNLS refers to the nuclear localization sequence having the sequence KRTADGSEFEPKKKRKV (SEQ ID NO: 740). Constructs are shown from N-terminus (left) to C-terminus (right).

1031<tables id="TABLE-US-00181" num="00181"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="56pt" align="left" /><colspec colname="2" colwidth="161pt" align="left" /><tbody valign="top"><row><entry>Construct 10:</entry><entry>Apobec-SGGSSGGSXTENSGGSSGGS-LbCpf1-</entry></row><row><entry></entry></row><row><entry /><entry>SGGSGGSGGS-UGI-SGGSGGSGGS-UGI-SGGS-</entry></row><row><entry></entry></row><row><entry /><entry>BPNLS</entry></row><row><entry></entry></row><row><entry>Construct 11:</entry><entry>Apobec-SGGSSGGSXTENSGGSSGGS-LbCpf1-</entry></row><row><entry></entry></row><row><entry /><entry>SGGSGGSGGS-UGI-SGGS-BPNLS</entry></row><row><entry></entry></row><row><entry>Construct 12:</entry><entry>UGI-SGGSGGSGGS-Apobec-SGGSSGGSXTENSG</entry></row><row><entry></entry></row><row><entry /><entry>GSSGGS-LbCpf1-SGGS-BPNLS</entry></row><row><entry></entry></row><row><entry>Construct 13:</entry><entry>Apobec-SGGSGGSGGS-UGI-SGGSSGGSXTENSG</entry></row><row><entry></entry></row><row><entry /><entry>GSSGGS-LbCpf1-SGGS-BPNLS</entry></row><row><entry></entry></row><row><entry>Construct 14:</entry><entry>LbCpf1-SGGSGGSGGS-UGI-SGGSGGSGGS-</entry></row><row><entry></entry></row><row><entry /><entry>Apobec-SGGS-BPNLS</entry></row><row><entry></entry></row><row><entry>Construct 15:</entry><entry>LbCpf1-SGGSGGSGGS-Apobec-SGGSGGSGGS-</entry></row><row><entry></entry></row><row><entry /><entry>UGI-SGGS-BPNLS</entry></row></tbody></tgroup></table></tables>

1032The common guide backbone (sgRNA) used in the experiments is GTAATTTCTACTAAGTGTAGAT (SEQ ID NO: 741)[guide sequence]TTTTTTT, wherein each of the Ts of SEQ ID NO: 741 are uracil (U), and where the guide sequence that targets the construct to a specific nucleotide sequence is shown between brackets. In some embodiments, any of the constructs provided herein are complexed with a sgRNA that comprises the backbone sequence of GTAATTTCTACTAAGTGTAGAT(SEQ ID NO: 741), wherein each of the Ts of SEQ ID NO: 741 are uracil (U). In some embodiments, any of the guide RNAs provided herein comprise a guide sequence comprising 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides that are perfectly complementary to a sequence, e.g., a target DNA sequence. In the experiments performed, the guide sequences tested are shown below:

1033<tables id="TABLE-US-00182" num="00182"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="42pt" align="left" /><colspec colname="2" colwidth="105pt" align="left" /><colspec colname="3" colwidth="70pt" align="left" /><tbody valign="top"><row><entry>EMX23:</entry><entry>TACTTTGTCCTCCGGTTCTGGAA</entry><entry>(SEQ ID NO: 742)</entry></row><row><entry></entry></row><row><entry>EMX20:</entry><entry>TACTTTGTCCTCCGGTTCTG</entry><entry>(SEQ ID NO: 743)</entry></row><row><entry></entry></row><row><entry>EMX19:</entry><entry>TACTTTGTCCTCCGGTTCT</entry><entry>(SEQ ID NO: 744)</entry></row><row><entry></entry></row><row><entry>EMX18:</entry><entry>TACTTTGTCCTCCGGTTC</entry><entry>(SEQ ID NO: 745)</entry></row><row><entry></entry></row><row><entry>EMX17:</entry><entry>TACTTTGTCCTCCGGTT</entry><entry>(SEQ ID NO: 746)</entry></row><row><entry></entry></row><row><entry>Hek2_23:</entry><entry>CAGCCCGCTGGCCCTGTAAAGGA</entry><entry>(SEQ ID NO: 747)</entry></row><row><entry></entry></row><row><entry>Hek2_20:</entry><entry>CAGCCCGCTGGCCCTGTAAA</entry><entry>(SEQ ID NO: 748)</entry></row><row><entry></entry></row><row><entry>Hek2_19:</entry><entry>CAGCCCGCTGGCCCTGTAA</entry><entry>(SEQ ID NO: 749)</entry></row><row><entry></entry></row><row><entry>Hek2_18:</entry><entry>CAGCCCGCTGGCCCTGTA</entry><entry>(SEQ ID NO: 750)</entry></row><row><entry></entry></row><row><entry>Hek2_17:</entry><entry>CAGCCCGCTGGCCCTGT</entry><entry>(SEQ ID NO: 751)</entry></row></tbody></tgroup></table></tables>

1034The data demonstrating the C to T base pair editing percentage using various constructs and target sequences is shown in <figref idref="DRAWINGS">FIGS. 153-159</figref>, and the editing percentage values (after adjustment based on indel count), and the percentage of indels for the experiments are shown in <figref idref="DRAWINGS">FIGS. 160-166</figref>.

0000Supplementary Sequences

1035Amino Acid Sequences of CDA1-BE3, AID-BE3, BE4, and SaBE4 fusion proteins. <u style="single">CDA1</u>-XTEN-<u style="single">dCas9</u>-<u style="single">UGI</u>-NLS primary sequence (CDA1-BE3):

1036<tables id="TABLE-US-00183" num="00183"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 165)</entry></row><row><entry><u style="single"><i>MTDAEYVRIHEKLDIYTFKK</i><i>Q</i><i>FFNNKKSVSHRCYVLFELKRRGERRACFW</i></u></entry></row><row><entry></entry></row><row><entry><u style="single"><i>GYAVNKP</i><i>Q</i><i>SGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADC</i></u></entry></row><row><entry></entry></row><row><entry><u style="single"><i>AEKILEWYNQELRGNGHTLKIWACKLYYEKNARN</i><i>Q</i><i>IGLWNLRDNGVGLNV</i></u></entry></row><row><entry></entry></row><row><entry><u style="single"><i>MVSEHY</i><i>Q</i><i>CCRKIFI</i><i>Q</i><i>SSHN</i><i>Q</i><i>LNENRWLEKTLKRAEKRRSELSIMIQVKIL</i></u></entry></row><row><entry></entry></row><row><entry><u style="single"><i>HTTKSPAV</i></u><b>SGSETPGTSESATPES</b><u style="single">DKKYSIGLAIGTNSVGWAVITDEYKV</u></entry></row><row><entry></entry></row><row><entry><u style="single">PSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKN</u></entry></row><row><entry></entry></row><row><entry><u style="single">RICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVA</u></entry></row><row><entry></entry></row><row><entry><u style="single">YHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPD</u></entry></row><row><entry></entry></row><row><entry><u style="single">NSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIA</u></entry></row><row><entry></entry></row><row><entry><u style="single">QLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN</u></entry></row><row><entry></entry></row><row><entry><u style="single">LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE</u></entry></row><row><entry></entry></row><row><entry><u style="single">HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIK</u></entry></row><row><entry></entry></row><row><entry><u style="single">PILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQE</u></entry></row><row><entry></entry></row><row><entry><u style="single">DFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN</u></entry></row><row><entry></entry></row><row><entry><u style="single">FEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKV</u></entry></row><row><entry></entry></row><row><entry><u style="single">KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS</u></entry></row><row><entry></entry></row><row><entry><u style="single">VETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFE</u></entry></row><row><entry></entry></row><row><entry><u style="single">DREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK</u></entry></row><row><entry></entry></row><row><entry><u style="single">TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL</u></entry></row><row><entry></entry></row><row><entry><u style="single">AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNS</u></entry></row><row><entry></entry></row><row><entry><u style="single">RERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE</u></entry></row><row><entry></entry></row><row><entry><u style="single">LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVK</u></entry></row><row><entry></entry></row><row><entry><u style="single">KMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI</u></entry></row><row><entry></entry></row><row><entry><u style="single">TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE</u></entry></row><row><entry></entry></row><row><entry><u style="single">INNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ</u></entry></row><row><entry></entry></row><row><entry><u style="single">EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG</u></entry></row><row><entry></entry></row><row><entry><u style="single">RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD</u></entry></row><row><entry></entry></row><row><entry><u style="single">PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN</u></entry></row><row><entry></entry></row><row><entry><u style="single">PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA</u></entry></row><row><entry></entry></row><row><entry><u style="single">LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS</u></entry></row><row><entry></entry></row><row><entry><u style="single">KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF</u></entry></row><row><entry></entry></row><row><entry><u style="single">DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD</u>SGGS<b>TNLSD</b></entry></row><row><entry></entry></row><row><entry><b>IIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVM</b></entry></row><row><entry></entry></row><row><entry>LLTSDAPEYKPWALVI<b>QDSNGENKIKML</b>SGGS<i>PKKKRKV</i></entry></row></tbody></tgroup></table></tables><br /><u style="single">AID</u>-XTEN-<u style="single">dCas9</u>-<u style="single">UGI</u>-NLS Primary Sequence (AID-BE3):

1037<tables id="TABLE-US-00184" num="00184"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 166)</entry></row><row><entry><u style="single"><i>MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLR</i></u></entry></row><row><entry></entry></row><row><entry><u style="single"><i>NKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG</i></u></entry></row><row><entry></entry></row><row><entry><u style="single"><i>NPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNT</i></u></entry></row><row><entry></entry></row><row><entry><u style="single"><i>FVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL</i></u><b>SG</b></entry></row><row><entry></entry></row><row><entry><b>SETPGTSESATPES</b><u style="single">DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGN</u></entry></row><row><entry></entry></row><row><entry><u style="single">TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFS</u></entry></row><row><entry></entry></row><row><entry><u style="single">NEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYH</u></entry></row><row><entry></entry></row><row><entry><u style="single">LRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQ</u></entry></row><row><entry></entry></row><row><entry><u style="single">LVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGL</u></entry></row><row><entry></entry></row><row><entry><u style="single">FGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA</u></entry></row><row><entry></entry></row><row><entry><u style="single">DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKA</u></entry></row><row><entry></entry></row><row><entry><u style="single">LVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTE</u></entry></row><row><entry></entry></row><row><entry><u style="single">ELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNR</u></entry></row><row><entry></entry></row><row><entry><u style="single">EKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGAS</u></entry></row><row><entry></entry></row><row><entry><u style="single">AQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKP</u></entry></row><row><entry></entry></row><row><entry><u style="single">AFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRF</u></entry></row><row><entry></entry></row><row><entry><u style="single">NASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLK</u></entry></row><row><entry></entry></row><row><entry><u style="single">TYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDG</u></entry></row><row><entry></entry></row><row><entry><u style="single">FANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI</u></entry></row><row><entry></entry></row><row><entry><u style="single">LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEG</u></entry></row><row><entry></entry></row><row><entry><u style="single">IKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYD</u></entry></row><row><entry></entry></row><row><entry><u style="single">VDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLL</u></entry></row><row><entry></entry></row><row><entry><u style="single">NAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDS</u></entry></row><row><entry></entry></row><row><entry><u style="single">RMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA</u></entry></row><row><entry></entry></row><row><entry><u style="single">YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF</u></entry></row><row><entry></entry></row><row><entry><u style="single">FYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVL</u></entry></row><row><entry></entry></row><row><entry><u style="single">SMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSP</u></entry></row><row><entry></entry></row><row><entry><u style="single">TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGY</u></entry></row><row><entry></entry></row><row><entry><u style="single">KEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLY</u></entry></row><row><entry></entry></row><row><entry><u style="single">LASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANL</u></entry></row><row><entry></entry></row><row><entry><u style="single">DKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYT</u></entry></row><row><entry></entry></row><row><entry><u style="single">STKEVLDATLIHQSITGLYETRIDLSQLGGD</u>SGGS<u style="single"><b>TNLSDHEKETGKQLV</b></u></entry></row><row><entry></entry></row><row><entry><u style="single"><b>IQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKP</b></u></entry></row><row><entry></entry></row><row><entry><u style="single"><b>WALVIQIDSNGENKIKML</b></u>SGGS<i>PKKKRKV</i></entry></row></tbody></tgroup></table></tables><br /><u style="single">rAPOBEC1</u>-linker-<u style="single">dCas9</u>-<u style="single">UGI</u>-<u style="single">UGI</u>-NLS Primary Sequence (BE4):

1038<tables id="TABLE-US-00185" num="00185"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 167)</entry></row><row><entry><u style="single"><i>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</i></u></entry></row><row><entry></entry></row><row><entry><u style="single"><i>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</i></u></entry></row><row><entry></entry></row><row><entry><u style="single"><i>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</i></u></entry></row><row><entry></entry></row><row><entry><u style="single"><i>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCHLGLPPCLNILRRKOP</i></u></entry></row><row><entry></entry></row><row><entry><u style="single"><i>QLTFFTIALQSCHYQRLPPHILWATGLK</i></u><b>SGGSSGGSSGSETPGTSESATP</b></entry></row><row><entry></entry></row><row><entry><b>ESSGGSSGGS</b><u style="single">DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRH</u></entry></row><row><entry></entry></row><row><entry><u style="single">SIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMA</u></entry></row><row><entry></entry></row><row><entry><u style="single">KVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKK</u></entry></row><row><entry></entry></row><row><entry><u style="single">LVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQT</u></entry></row><row><entry></entry></row><row><entry><u style="single">YNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL</u></entry></row><row><entry></entry></row><row><entry><u style="single">IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFL</u></entry></row><row><entry></entry></row><row><entry><u style="single">AAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ</u></entry></row><row><entry></entry></row><row><entry><u style="single">QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLV</u></entry></row><row><entry></entry></row><row><entry><u style="single">KLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE</u></entry></row><row><entry></entry></row><row><entry><u style="single">KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSF</u></entry></row><row><entry></entry></row><row><entry><u style="single">IERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS</u></entry></row><row><entry></entry></row><row><entry><u style="single">GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASL</u></entry></row><row><entry></entry></row><row><entry><u style="single">GTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAH</u></entry></row><row><entry></entry></row><row><entry><u style="single">LFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR</u></entry></row><row><entry></entry></row><row><entry><u style="single">NFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTV</u></entry></row><row><entry></entry></row><row><entry><u style="single">KVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEL</u></entry></row><row><entry></entry></row><row><entry><u style="single">GSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI</u></entry></row><row><entry></entry></row><row><entry><u style="single">VPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKL</u></entry></row><row><entry></entry></row><row><entry><u style="single">ITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNT</u></entry></row><row><entry></entry></row><row><entry><u style="single">KYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA</u></entry></row><row><entry></entry></row><row><entry><u style="single">VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN</u></entry></row><row><entry></entry></row><row><entry><u style="single">IMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQ</u></entry></row><row><entry></entry></row><row><entry><u style="single">VNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAY</u></entry></row><row><entry></entry></row><row><entry><u style="single">SVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVK</u></entry></row><row><entry></entry></row><row><entry><u style="single">KDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH</u></entry></row><row><entry></entry></row><row><entry><u style="single">YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVL</u></entry></row><row><entry></entry></row><row><entry><u style="single">SAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKE</u></entry></row><row><entry></entry></row><row><entry><u style="single">VLDATLIHQSITGLYETRIDLSQLGGD</u>SGGSGGSGGS<u style="single"><b>TNLSDITEKETGK</b></u></entry></row><row><entry></entry></row><row><entry><u style="single"><b>QLVICIESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAP</b></u></entry></row><row><entry></entry></row><row><entry><u style="single"><b>EYKPWALVICIDSNGENKIKML</b></u>SGGSGGSGGS <img file="US11268082B2_D0268.tif" /><img file="US11268082B2_D0269.tif" /></entry></row><row><entry></entry></row><row><entry><img file="US11268082B2_D0270.tif" /><img file="US11268082B2_D0271.tif" /><img file="US11268082B2_D0272.tif" /><img file="US11268082B2_D0273.tif" /><img file="US11268082B2_D0274.tif" /><img file="US11268082B2_D0275.tif" /></entry></row><row><entry></entry></row><row><entry><img file="US11268082B2_D0276.tif" /><img file="US11268082B2_D0277.tif" /> SGGS<i>PKKKRK</i></entry></row></tbody></tgroup></table></tables><br /><u style="single">rAPOBEC1</u>-linker-<u style="single">SaCas9d</u>-<u style="single">UGI</u>-<u style="single">UGI</u>-NLS Primary Sequence (SaBE4):

1039<tables id="TABLE-US-00186" num="00186"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><tbody valign="top"><row><entry>(SEQ ID NO: 168)</entry></row><row><entry><u style="single"><i>MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI</i></u></entry></row><row><entry></entry></row><row><entry><u style="single"><i>WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI</i></u></entry></row><row><entry></entry></row><row><entry><u style="single"><i>TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG</i></u></entry></row><row><entry></entry></row><row><entry><u style="single"><i>YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCHLGLPPCLNILRRKQP</i></u></entry></row><row><entry></entry></row><row><entry><u style="single"><i>QLTFFTIALQSCHYQRLPPHILWATGLK</i></u><b>SGGSSGGSSGSETPGTSESATP</b></entry></row><row><entry></entry></row><row><entry><b>ESSGGSSGGSG</b><u style="single">KRNYILGLAIGITSVGYGIIDYETRDVIDAGVRLFKEAN</u></entry></row><row><entry></entry></row><row><entry><u style="single">VENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYE</u></entry></row><row><entry></entry></row><row><entry><u style="single">ARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISR</u></entry></row><row><entry></entry></row><row><entry><u style="single">NSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAY</u></entry></row><row><entry></entry></row><row><entry><u style="single">HQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYF</u></entry></row><row><entry></entry></row><row><entry><u style="single">PEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQ</u></entry></row><row><entry></entry></row><row><entry><u style="single">KKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKE</u></entry></row><row><entry></entry></row><row><entry><u style="single">IIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYT</u></entry></row><row><entry></entry></row><row><entry><u style="single">GTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTL</u></entry></row><row><entry></entry></row><row><entry><u style="single">VDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKM</u></entry></row><row><entry></entry></row><row><entry><u style="single">INEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEA</u></entry></row><row><entry></entry></row><row><entry><u style="single">IPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQ</u></entry></row><row><entry></entry></row><row><entry><u style="single">YLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKD</u></entry></row><row><entry></entry></row><row><entry><u style="single">FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKF</u></entry></row><row><entry></entry></row><row><entry><u style="single">KKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAES</u></entry></row><row><entry></entry></row><row><entry><u style="single">MPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYS</u></entry></row><row><entry></entry></row><row><entry><u style="single">TRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKL</u></entry></row><row><entry></entry></row><row><entry><u style="single">KLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAH</u></entry></row><row><entry></entry></row><row><entry><u style="single">LDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYY</u></entry></row><row><entry></entry></row><row><entry><u style="single">EVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN</u></entry></row><row><entry></entry></row><row><entry><u style="single">RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYE</u></entry></row><row><entry></entry></row><row><entry><u style="single">VKSKKHPQIIKKG</u>GSPKKKRKVSSDYKDHDGDYKDHDIDYKDDDDKSGGS</entry></row><row><entry></entry></row><row><entry>GGSGGS<u style="single"><b>TNLSDHEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA</b></u></entry></row><row><entry></entry></row><row><entry><u style="single"><b>YDESTDENVMLLTSDAPEYKPWALVICIDSNGENKIKML</b></u>SGGSGGSGGS</entry></row><row><entry></entry></row><row><entry><img file="US11268082B2_D0278.tif" /><img file="US11268082B2_D0279.tif" /><img file="US11268082B2_D0280.tif" /><img file="US11268082B2_D0281.tif" /><img file="US11268082B2_D0282.tif" /><img file="US11268082B2_D0283.tif" /></entry></row><row><entry></entry></row><row><entry><img file="US11268082B2_D0284.tif" /><img file="US11268082B2_D0285.tif" /><img file="US11268082B2_D0286.tif" /><img file="US11268082B2_D0287.tif" /><img file="US11268082B2_D0288.tif" /> SGGS<i>PKKKRKV</i></entry></row></tbody></tgroup></table></tables>

References for Example 17

0000<ul id="ul0009" list-style="none"><li id="ul0009-0001" num="1040">1. A. C. Komor, Y. B. Kim, M. S. Packer, J. A. Zuris, D. R. Liu, Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424 (2016).</li><li id="ul0009-0002" num="1041">2. G. T. Hess et al., Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat Methods 13, 1036-1042 (2016).</li><li id="ul0009-0003" num="1042">3. K. Kim et al., Highly efficient RNA-guided base editing in mouse embryos. Nat Biotechnol 35, 435-437 (2017).</li><li id="ul0009-0004" num="1043">4. Y. B. Kim et al., Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat Biotechnol 35, 371-376 (2017).</li><li id="ul0009-0005" num="1044">5. C. Kuscu et al., CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations. Nat Meth advance online publication, (2017).</li><li id="ul0009-0006" num="1045">6. J. Li, Y. Sun, J. Du, Y. Zhao, L. Xia, Generation of Targeted Point Mutations in Rice by a Modified CRISPR/Cas9 System. Mol Plant 10, 526-529 (2017).</li><li id="ul0009-0007" num="1046">7. Y. Lu, J. K. Zhu, Precise Editing of a Target Base in the Rice Genome Using a Modified CRISPR/Cas9 System. Mol Plant 10, 523-525 (2017).</li><li id="ul0009-0008" num="1047">8. Y. Ma et al., Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat Methods 13, 1029-1035 (2016).</li><li id="ul0009-0009" num="1048">9. K. Nishida et al., Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, (2016).</li><li id="ul0009-0010" num="1049">10. H. A. Rees et al., Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun 8, 15790 (2017).</li><li id="ul0009-0011" num="1050">11. L. Yang et al., Engineering and optimising deaminase fusions for genome editing. Nat Commun 7, 13330 (2016).</li><li id="ul0009-0012" num="1051">12. Y. Zong et al., Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat Biotechnol 35, 438-440 (2017).</li><li id="ul0009-0013" num="1052">13. Z. Shimatani et al., Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat Biotech 35, 441-443 (2017).</li><li id="ul0009-0014" num="1053">14. A. C. Komor, A. H. Badran, D. R. Liu, CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes. Cell 168, 20-36 (2017).</li><li id="ul0009-0015" num="1054">15. A. J. Davis, D. J. Chen, DNA double strand break repair via non-homologous end-joining. Translational cancer research 2, 130-143 (2013).</li><li id="ul0009-0016" num="1055">16. M. M. Vilenchik, A. G. Knudson, Endogenous DNA double-strand breaks: Production, fidelity of repair, and induction of cancer. Proceedings of the National Academy of Sciences 100, 12871-12876 (2003).</li><li id="ul0009-0017" num="1056">17. F. Liang, M. Han, P. J. Romanienko, M. Jasin, Homology-directed repair is a major doublestrand break repair pathway in mammalian cells. Proceedings of the National Academy of Sciences 95, 5172-5177 (1998).</li><li id="ul0009-0018" num="1057">18. Y. Miyaoka et al., Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Scientific Reports 6, 23549 (2016).</li><li id="ul0009-0019" num="1058">19. M. M. Jore et al., Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol 18, 529-536 (2011).</li><li id="ul0009-0020" num="1059">20. L. H. Pearl, Structure and function in the uracil-DNA glycosylase superfamily. Mutation Research/DNA Repair 460, 165-181 (2000).</li><li id="ul0009-0021" num="1060">21. W. Tang, J. H. Hu, D. R. Liu, Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation. 8, 15939 (2017).</li><li id="ul0009-0022" num="1061">22. G. Saraconi, F. Severi, C. Sala, G. Mattiuz, S. G. Conticello, The RNA editing enzyme APOBEC1 induces somatic mutations and a compatible mutational signature is present in esophageal adenocarcinomas. Genome Biology 15, 417 (2014).</li><li id="ul0009-0023" num="1062">23. R. M. Kohli et al., Local Sequence Targeting in the AID/APOBEC Family Differentially Impacts Retroviral Restriction and Antibody Diversification. Journal of Biological Chemistry 285, 40956-40964 (2010).</li><li id="ul0009-0024" num="1063">24. L. Chelico, P. Pham, M. F. Goodman, Stochastic properties of processive cytidine DNA deaminases AID and APOBEC3G. Philosophical Transactions of the Royal Society B: Biological Sciences 364, 583-593 (2009).</li><li id="ul0009-0025" num="1064">25. E. A. Kouzminova, A. Kuzminov, Patterns of chromosomal fragmentation due to uracil-DNA incorporation reveal a novel mechanism of replication-dependent double-stranded breaks. Molecular Microbiology 68, 202-215 (2008).</li><li id="ul0009-0026" num="1065">26. F. A. Ran et al., In vivo genome editing using <i>Staphylococcus aureus </i>Cas9. Nature 520, 186-191 (2015).</li><li id="ul0009-0027" num="1066">27. F. d. A. di Fagagna, G. R. Weller, A. J. Doherty, S. P. Jackson, The Gam protein of bacteriophage Mu is an orthologue of eukaryotic Ku. EMBO Reports 4, 47-52 (2003).</li><li id="ul0009-0028" num="1067">28. C. Shee et al., Engineered proteins detect spontaneous DNA breakage in human and bacterial cells. eLife 2, e01222 (2013).</li></ul>

EQUIVALENTS AND SCOPE

1068Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

1069Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context. The disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

1070It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

1071Where elements are presented as lists, e.g., in Markush group format, it is to be understood that every possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where an embodiment, product, or method is referred to as comprising particular elements, features, or steps, embodiments, products, or methods that consist, or consist essentially of, such elements, features, or steps, are provided as well. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

1072Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range. In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.